Multispecific antigen-binding molecules

ABSTRACT

Novel antigen-binding molecules are provided, with the ability to target different antigens with different valency, e.g. one antigen monovalently and another antigen bivalently.

TECHNICAL FIELD

The present disclosure provides novel antigen-binding molecules capableof simultaneously binding to at least two different antigens. Theability to target two different antigens with different valency (e.g.one antigen monovalently and one antigen bivalently) is a particularuseful aspect of the antigen-binding molecules disclosed herein. Thenovel molecules described herein preferably utilize heterodimeric Fcregions of an immunoglobulin. Methods of producing and using such novelantigen-binding molecules and compositions comprising such, particularlyfor therapeutic purposes are also described.

BACKGROUND

Multispecific antigen-binding molecules, such as bispecific antibodies,capable of binding to two or more antigens are of great interest fortherapeutic applications, as they allow for the simultaneous binding andinactivation of two or more target antigens, and as such represent analternative approach to conventional combination therapies. However, formany antigens that are attractive as co-targets for such multispecificformats, the preferred binding to at least one antigen is monovalentrather than bivalent.

Bivalent binding of regular immunoglobulin antibodies has been found tocrosslink certain cell surface receptors and thereby mimic the effect ofthe natural ligand. Cross-linking can lead to receptor activation (e.g.receptor phosphorylation). In contrast, monovalent binding (such as ofFabs derived from the same antibody) does not lead to receptorcross-linking and, if the appropriate epitope is targeted, prevent thenatural ligand from binding. Thus, while bivalent antigen-binding mightresult in an agonistic activity, monovalent binding to the same antigenmight result in an antagonistic activity. Examples of such receptors arethe insulin receptor (Kahn et al., Proc Natl Acad Sci USA. (1978)75:4209-13), the EGF receptor (Schreiber et al., J Biol Chem. (1983)258:846-53), the EPO receptor (Schneider et al, Blood (1997) 89:473-82),the GH receptor (Wan et al., Mol Endocrinol. (2003) 17:2240-50) or thebeta2-Adrenoceptor (Mijares et al., Mol Pharmacol. (2000) 58:373-9).

Other exemplary antigens for which it may be therapeutically beneficialor necessary to co-engage monovalently include members of the T-cellreceptor complex, such as CD3, the low affinity Fc gamma receptors(FcyRs), toll-like receptors (TLRs), cytokines, chemokines, cytokinereceptors, chemokine receptors or receptor-tyrosine kinases (RTKs).

A large number of multispecific antigen-binding formats were developedin the recent years, including tetravalent IgG-single-chain variablefragment (scFv) fusions (see e.g. Coloma & Morrison, Nat Biotechnol 15,159-163 (1997)), tetravalent IgG-like dual-variable domain (DVD)antibodies (Wu et al., Nat Biotechnol 25, 1290-1297 (2007)), or bivalentrat/mouse hybrid bispecific IgGs (see e.g. Lindhofer et al., J Immunol155, 219-225 (1995)).

However, a disadvantage of such IgG based approaches is that they bindto the co-targeted antigen in a multivalent (e.g. bivalent) fashion,thus leading to a potential non-specific activation and associatedside-effects. The production of these IgG-based multispecific constructsis also a major hurdle, as the homodimerization of antibody heavy chainsand/or the mispairing of antibody heavy and light chains of differentspecificities upon co-expression decreases the yield of the correctlyassembled construct and results in a number of non-functional sideproducts from which the desired construct may be difficult to separate.

On the other hand, several multispecific antigen-binding formats,wherein an antibody core structure (IgA, IgD, IgE, IgG or IgM) is nolonger maintained were developed. Examples include diabodies (see e.g.Holliger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1995)), tandemscFv molecules (see e.g. Bargou et al., Science 321, 974-977 (2008)),and various derivatives thereof. However, antigen-binding formatslacking the IgG core structure and being capable of providing monovalentbinding to at least one antigen are often disadvantageous because oftheir poor biophysical and pharmacokinetic properties, such as shorthalf-life and their incapability to mediate effector function (such asADCC or CDC).

WO 2016/086189 discloses various multispecific bi- and trivalentantibody formats based on a full IgG molecule wherein either one Fab armof the IgG molecule is replaced by a single-chain Fv domain (“bottleopener” format) or wherein an additional Fv or single-chain Fv domain isfused to the C-terminus of one or both heavy chains of the IgG molecule.One additional theoretical described antibody format refers to the“central-Fv” format which incorporates an additional Fv domain betweenthe two Fab arms and the Fc region of an IgG in order to form a thirdantigen binding site. However, the application neither providesexperimental evidence that the described construct does actuallyfunction nor does it suggests to combine the specific Fc modificationsutilized in the antigen-binding molecules of the present disclosure,responsible for efficient heterodimerization of the two Fc regionsubunits and/or abolished effector function.

The presently disclosed novel antigen-binding molecules solve theaforementioned shortcomings of the IgG- and the non-IgG basedantigen-binding molecules by introducing a format that allows for thesimultaneous bivalent and monovalent co-engagement of different antigenswith all the desirable properties provided by a regular full-lengthimmunoglobulin and which are easy to purify from the culture supernatantof respective production cell lines.

SUMMARY

The present disclosure pertains to novel multispecific antigen-bindingmolecules, which allow for monovalent binding to at least one antigen,whilst retaining the properties of a regular immunoglobulin molecule inrespect of size and presence of an Fc region.

In an embodiment, an antigen-binding molecule according to the presentdisclosure allows for trivalent binding to one antigen. In such anembodiment, the antigen-binding molecule comprises at least three Fvregions, wherein each Fv region binds to the same antigen. In such anembodiment, the antigen-binding molecule according to the presentdisclosure refers to a trivalent monospecific antigen-binding molecule.

In an embodiment, an antigen-binding molecule according to the presentdisclosure allows for a monovalent binding to three different antigens.In such an embodiment, the antigen-binding molecule comprises at leastthree Fv regions, wherein each of the three Fv regions binds to onedifferent antigen. In such an embodiment, the antigen-binding moleculeaccording to the present disclosure refers to a trivalent trispecificantigen-binding molecule.

In a preferred embodiment, an antigen-binding molecule according to thepresent disclosure allows for a bivalent binding to one antigen andmonovalent binding to a second antigen. In such an embodiment, theantigen-binding molecule comprises at least three Fv regions, whereintwo of the three Fv regions binds to one of the two target antigens andthe third Fv region binds to the other target antigen. In such apreferred embodiment, an antigen-binding molecule according to thepresent disclosure refers to a trivalent bispecific antigen-bindingmolecule.

Accordingly, in an embodiment, the present disclosure provides anantigen-binding molecule comprising

-   -   a) a first Fab comprising a first Fv region, which specifically        binds to a first antigen, b) a second Fv region which        specifically binds to a second antigen and    -   c) a second Fab comprising a third Fv region, which specifically        binds to a third antigen, and    -   d) a Fc region composed of a first and second Fc region subunit;        wherein        -   I. the C-terminus of the heavy or light chain of the first            Fab is fused to the N-terminus of the VH or VL of the second            Fv region, and wherein        -   II. the C-terminus of the VH or VL of the second Fv region            is fused to the N-terminus of the first Fc region subunit            and the N-terminus of the second Fc domain subunit is fused            to the C-terminus of the complementary variable domain of            the second Fv region, and wherein        -   III. the C-terminus of the heavy or light chain of the            second Fab is fused to the N-terminus of the VH or VL of the            second Fv region with the proviso that the first and second            Fab are fused to distinct variable domains of the second Fv            region, and wherein        -   IV. in the CH3 domain of first Fc region subunit, the            threonine residue at position 366 is replaced with a            tryptophan residue (T366W) and the serine residue at            position 354 is replaced with a cysteine residue (S354C) and            in the CH3 domain of the second Fc region subunit the            tyrosine residue at position 407 is replaced with a valine            residue (Y407V), the threonine residue at position 366 is            replaced with a serine residue (T366S), the leucine residue            at position 368 is replaced with an alanine residue (L368A)            and the tyrosine residue at position 349 is replaced by a            cysteine residue (Y349C) with numbering according EU index.

In an embodiment, the third antigen is identical to the first or thesecond antigen. In an embodiment, the third antigen is identical to thefirst antigen. In an embodiment, the first Fab is identical to thesecond Fab.

In an embodiment, the second Fab is fused to the second Fv region. In anembodiment, the C-terminus of the second Fab is fused to the N-terminusof the second Fv region. In an embodiment, the C-terminus of the heavyor light chain of the second Fab is fused to the N-terminus of the VH orVL of the second Fv region with the proviso that the first and secondFab are fused to distinct variable domains of the second Fv region. Inan embodiment, the C-terminus of the CH1 or CL of the second Fab isfused to the N-terminus of the VH or VL of the second Fv region with theproviso that the first and second Fab are fused to distinct variabledomains of the second Fv region.

In an embodiment, each fusion occurs via a linker. In an embodiment,each fusion occurs via a peptide linker. In an embodiment, the peptidelinkers are identical or different. In an embodiment, each fusion occursvia a peptide linker each having a length of at least 1 amino acidsresidue. In an embodiment, each fusion occurs via a peptide linker eachhaving a length of at least 5 amino acids residues. In an embodiment,the peptide linkers are of identical length. In an embodiment, thepeptide linkers are of different length. In an embodiment, the first Fabis fused to the second Fv region via a peptide linker.

In an embodiment, the C-terminus of the heavy or light chain of thefirst Fab is fused to the N-terminus of the VH or VL of the second Fvregion via a peptide linker. In an embodiment, the C-terminus of the CH1or CL of the first Fab is fused to the N-terminus of the VH or VL of thesecond Fv region via a peptide linker.

In an embodiment, the second Fv region is fused to the Fc region viapeptide linkers. In an embodiment, the second Fv region is fused to theFc region via two peptide linkers. In an embodiment, the C-terminus ofthe VH or VL of the second Fv region is fused to the N-terminus of thefirst Fc region subunit via a first peptide linker and the N-terminus ofthe second Fc region subunit is fused to the C-terminus of thecomplementary variable domain of the second Fv region via a secondpeptide linker. In an embodiment, the first and second peptide linkerare different. In an embodiment, the first and second peptide linker areidentical.

In an embodiment, the first and second peptide linker are linked via onemore interchain disulfide bridges. In an embodiment, the first andsecond peptide linker comprises one or more cysteine residues allowingfor the formation of one or more interchain disulfide bridges betweenthe first and second peptide linker. In an embodiment, the first andsecond peptide linker comprises one or more cysteine residues allowingfor the formation of one or more interchain disulfide bridges betweenthe first and second peptide linker resulting in a disulfide bridgestabilized dimeric peptide linker. In an embodiment, the first andsecond peptide linker is derived from an immunoglobulin hinge,preferably from an IgG hinge, preferably from a human IgG hinge,preferably a human IgG1 hinge of fragment thereof. In an embodiment, thefusion between the VH or VL of the second Fv region and the first orsecond Fc region subunit occurs via a peptide linker comprising an IgGhinge or a portion or fragment thereof. In an embodiment, the IgG hingeis a human IgG hinge. In an embodiment, the human IgG hinge is a humanIgG1 hinge. In an embodiment, the human IgG1 hinge comprises the aminosequence DKTHTCPPCP (SEQ ID NO: 13). In an embodiment, the fusionbetween the second Fv region and the Fc region occurs via an IgG hingeregion or part thereof.

In an embodiment, the first Fab, the second Fv region and the Fc regionare fused to each of their fusion partners via a peptide linker. In anembodiment, the peptide linkers are identical or different. In anembodiment, the peptide linkers are identical. In an embodiment, thepeptide linkers are different. In an embodiment, each of the peptidelinkers has a length of at least 1 amino acid residue. In an embodiment,each of the peptide linkers has a length of at least 5 amino acidresidues. In an embodiment, each of the peptide linkers has a length ofbetween 1 and 70 amino acid residues. In an embodiment, the peptidelinkers are of identical length. In an embodiment, the peptide linkersare of different length.

In an embodiment, the peptide linker is selected from the groupconsisting of but not limited to QPKAAP (SEQ ID NO: 12), ASTKGP (SEQ IDNO: 11), (G₄S)₃ (SEQ ID NO: 33), (GGS)₃ (SEQ ID NO: 10), DKTHTCPPCP (SEQID NO: 13), QPKAAPDKTHTCPPCP (SEQ ID NO: 15), and ASTKGPDKTHTCPPCP (SEQID NO: 14).

In an embodiment, the VH and the VL of the second Fv region areoptionally linked via an interchain disulfide bridge. In an embodiment,the VH and the VL of the second Fv region are linked via an interchaindisulfide bridge.

In an embodiment, the disulfide bridge is introduced between thefollowing positions with numbering according Kabat:

-   -   a. VH position 44 and VL position 100, and/or    -   b. VH position 105 and VL position 43 and/or    -   c. VH position 101 and VL position 100

In an embodiment, the disulfide bridge is introduced between thepositions with numbering according Kabat: VH position 44 and VL position100. In an embodiment, the disulfide bridge is introduced between thepositions with numbering according Kabat: VH position 105 and VLposition 43. In an embodiment, the disulfide bridge is introducedbetween the positions with numbering according Kabat: VH position 101and VL position 100. In an embodiment, the second Fab is fused to thesecond Fv region via a peptide linker

In an embodiment, the C-terminus of the heavy or light chain of thesecond Fab is fused to the N-terminus of the VH or VL of the second Fvregion via a peptide linker. In an embodiment, the C-terminus of the CH1or CL of the second Fab is fused to the N-terminus of the VH or VL ofthe second Fv region via a peptide linker. In an embodiment, theC-terminus of the CH1 or CL of the second Fab is fused to the N-terminusof the VH or VL of the second Fv region with the proviso that first andsecond Fab are fused to distinct variable domains of the second Fvregion and wherein each fusion occurs via a peptide linker.

In an embodiment, the antigen-binding molecule according to the presentdisclosure is composed of at least 4 polypeptides. In an embodiment, anantigen-binding molecule according to the present disclosure is composedof at least 4 polypeptides, wherein

-   -   a. a first polypeptide comprises the light or heavy chain of the        first Fab,    -   b, a second polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary light or heavy chain of the first Fab,        -   ii. the VH or VL of the second Fv region and        -   iii. the first or second Fc domain subunit    -   c. a third polypeptide comprises from its N-terminus to its        C-terminus        -   i. the light or heavy chain of the second Fab,        -   ii. the complementary VH or VL of the second Fv region and        -   iii. the complementary first or second Fc domain subunit.    -   d. a fourth polypeptide comprises the complementary light or        heavy chain of the second Fab.

In an embodiment, the first polypeptide is identical to the fourthpolypeptide. In an embodiment, the light chain of the first Fab isidentical to light chain of the second Fab. In an embodiment, the heavychain of the first Fab is identical to the heavy chain of the secondFab. In an embodiment, the heavy and light chain of the first and secondFab are identical. In an embodiment, an antigen-binding moleculeaccording to the present disclosure has a structure as depicted in FIG.1B.

In an embodiment, an antigen-binding molecule according to the presentdisclosure provides monovalent binding to the first, second antigen orthird antigen. In an embodiment, an antigen-binding molecule accordingto the present disclosure provides monovalent binding to the firstantigen. In an embodiment, an antigen-binding molecule according to thepresent disclosure provides monovalent binding to the second antigen. Inan embodiment, an antigen-binding molecule according to the presentdisclosure provides monovalent binding to the third antigen. In anembodiment, the third antigen is identical to the first or the secondantigen. In an embodiment, the third antigen is identical to the firstantigen. In an embodiment, an antigen-binding molecule according to thepresent disclosure provides monovalent binding to the first antigen andbivalent binding to the second antigen. In an embodiment, anantigen-binding molecule according to the present disclosure providesbivalent binding to the first antigen and monovalent binding to thesecond antigen.

In an embodiment, an antigen-binding molecule according to the presentdisclosure is a bispecific antigen-binding molecule. In an embodiment,an antigen-binding molecule according to the present disclosure is atrivalent bispecific antigen-binding molecule.

In an embodiment, the first or second antigen is a member of the T-cellreceptor complex. In an embodiment, the second antigen is a member ofthe T-cell receptor complex. In an embodiment, the first antigen is amember of the T-cell receptor complex. In an embodiment, the member ofthe T-cell receptor complex is CD3. In an embodiment, the first antigenis CD3. In an embodiment, the second antigen is CD3.

In an embodiment, an antigen-binding molecule according to the presentdisclosure provides bivalent binding to the first antigen and monovalentbinding to the second antigen, wherein the second antigen is CD3. In anembodiment, an antigen-binding molecule according to the presentdisclosure provides monovalent binding to the first antigen and bivalentbinding to the second antigen, wherein the first antigen is CD3.

In an embodiment, the present disclosure provides an antigen-bindingmolecule, wherein the Fc region comprises one or more amino acidmodifications promoting the association of the first and second Fcregion subunit. In an embodiment, the present disclosure provides anantigen-binding molecule, wherein each Fc region subunit comprises oneor more amino acid modifications promoting the association of the firstand second Fc region subunit. In an embodiment, each Fc region subunitcomprises a different amino acid modification, such that theheterodimeric Fc region is more stable than the homodimeric Fc region.In an embodiment, each Fc region subunit comprises a different aminoacid modification, such that the association of the first and second Fcregion subunit is promoted. In an embodiment, the Fc region is animmunoglobulin Fc region. In an embodiment, the immunoglobulin Fc regionis an IgG Fc region. In an embodiment, the IgG Fc region is a human IgGFc region. In an embodiment, the human IgG Fc region is a human IgG1region.

In an embodiment, in the CH3 domain of the first Fc region subunit thethreonine residue at position 366 is replaced with a tryptophan residue(T366W) and in the CH3 domain of the second Fc region subunit thetyrosine residue at position 407 is replaced with a valine residue(Y407V) with numbering according EU index. In an embodiment, in thesecond Fc region subunit, the threonine residue at position 366 isreplaced with a serine residue (T366S) and the leucine residue atposition 368 is replaced with an alanine residue (L368A) with numberingaccording EU index. In an embodiment, in the first Fc region subunit theserine residue at position 354 is replaced with a cysteine residue(S354C), and in the second Fc region subunit of the Fc region thetyrosine residue at position 349 is replaced by a cysteine residue(Y349C) with numbering according EU index.

In an embodiment, in the CH3 domain of first Fc region subunit, thethreonine residue at position 366 is replaced with a tryptophan residue(T366W) and the serine residue at position 354 is replaced with acysteine residue (S354C) and in the CH3 domain of the second Fc regionsubunit the tyrosine residue at position 407 is replaced with a valineresidue (Y407V), the threonine residue at position 366 is replaced witha serine residue (T366S), the leucine residue at position 368 isreplaced with an alanine residue (L368A) and the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C) with numberingaccording EU index.

In an embodiment, the Fc region is engineered to have an altered bindingaffinity to an Fc receptor and/or to C1q and/or to have an alteredeffector function when compared to the non-engineered Fc region. In anembodiment, the engineered Fc region has a higher binding affinity to anFc receptor and/or to C1q and/or has increased effector function whencompared to the non-engineered Fc region.

In an embodiment, the engineered Fc region has a lower binding affinityto an Fc receptor and/or to C1q and/or has reduced effector functionwhen compared to the non-engineered Fc region. In an embodiment, theengineered Fc region has substantially no binding affinity to an Fcreceptor and/or to C1q and/or has substantially no effector functionwhen compared to the non-engineered Fc region. In an embodiment, theengineered Fc region has no binding affinity to an Fc receptor and/or toC1q and/or has no effector function when compared to the non-engineeredFc region.

In an embodiment, the present disclosure provides an antigen-bindingmolecule, wherein in each Fc region subunit at least one of the 5 aminoacid residues in the positions corresponding to positions L234, L235,G237, A330, P331 with numbering according EU index in a human IgG1 aremutated. In an embodiment, the present disclosure provides anantigen-binding molecule, wherein in each Fc region subunit at least oneof the 5 amino acid residues in the positions corresponding to positionsL234, L235, G237, A330, P331 with numbering according EU index in ahuman IgG1 are mutated and wherein the engineered Fc region hassubstantially no binding affinity to an Fc receptor and/or to C1q and/orhas substantially no effector function when compared to thenon-engineered Fc region. In an embodiment, the present disclosureprovides an antigen-binding molecule, wherein in each Fc region subunitat least one of the 5 amino acid residues in the positions correspondingto positions L234, L235, G237, A330, P331 with numbering according EUindex in a human IgG1 are mutated to A, E, A, S, and S, respectively. Inan embodiment, the present disclosure provides an antigen-bindingmolecule, wherein in each Fc region subunit at least one of the 5 aminoacid residues in the positions corresponding to positions L234, L235,G237, A330, P331 with numbering according EU index in a human IgG1 aremutated to A, E, A, S, and S, respectively and wherein the engineered Fcregion has substantially no binding affinity to an Fc receptor and/or toC1q and/or has substantially no effector function when compared to thenon-engineered Fc region.

In an embodiment, the present disclosure provides an antigen-bindingmolecule, wherein in the Fc region subunit at least 5 amino acidresidues in the positions corresponding to positions L234, L235, G237,A330, P331 with numbering according EU index in a human IgG1 are mutatedto A, E, A, S, and S, respectively. In an embodiment, the presentdisclosure provides an antigen-binding molecule, wherein in the Fcregion subunit at least 5 amino acid residues in the positionscorresponding to positions L234, L235, G237, A330, P331 with numberingaccording EU index in a human IgG1 are mutated to A, E, A, S, and S,respectively and wherein the engineered Fc region has substantially nobinding affinity to an Fc receptor and/or to C1q and/or hassubstantially no effector function when compared to the non-engineeredFc region.

In an embodiment, the antigen-binding molecule according to the presentdisclosure is a polyclonal or monoclonal antigen-binding molecule. In anembodiment, the antigen-binding molecule according to the presentdisclosure is a monoclonal antigen-binding molecule.

In an embodiment, the antigen-binding molecule according to the presentdisclosure is an isolated antigen-binding molecule. In an embodiment,the antigen-binding molecule according to the present disclosure is arecombinant antigen-binding molecule. In an embodiment, theantigen-binding molecule according to the present disclosure is anisolated recombinant antigen-binding molecule.

In an embodiment, the present disclosure provides a nucleic acidcomposition comprising a nucleic acid sequence or a plurality of nucleicacid sequences encoding an antigen-binding molecule according to thepresent disclosure. In an embodiment, an antigen-binding moleculeaccording to the present disclosure is encoded by a nucleic acidcomposition according to the present disclosure. In an embodiment, thepresent disclosure provides a vector composition comprising a vector ora plurality of vectors comprising the nucleic acid composition accordingto the present disclosure. In an embodiment, the present disclosureprovides to a host cell comprising a vector composition according to thepresent disclosure or a nucleic acid composition according to thepresent disclosure encoding an antigen-binding molecule according to thepresent disclosure. In an embodiment, the present disclosure provides ahost cell comprising a nucleic acid composition according to the presentdisclosure or the vector composition according to the presentdisclosure. In an embodiment, the present disclosure provides a hostcell, wherein the host cell is a eukaryotic cell, particularly amammalian cell. In an embodiment, the present disclosure provides a hostcell, wherein the host cell is a eukaryotic cell. In an embodiment, thepresent disclosure provides a host cell, wherein the host cell is amammalian cell.

In an embodiment, the present disclosure provides a method of producingan antigen-binding molecule according to the present disclosure,comprising the steps of a) culturing a host cell according to thepresent disclosure under conditions suitable for the expression of theantigen-binding molecule and b) recovering the antigen-binding molecule.In an embodiment, the present disclosure provides an antigen-bindingmolecule produced by the method disclosed herein.

In an embodiment, the present disclosure provides a pharmaceuticalcomposition comprising an antigen-binding molecule according to thepresent disclosure and a pharmaceutically acceptable carrier. In anembodiment, the present disclosure provides a pharmaceutical compositioncomprising an antigen-binding molecule according to the presentdisclosure for use as a medicament. In an embodiment, the presentdisclosure provides an antigen-binding molecule according to the presentdisclosure or a pharmaceutical composition according to the presentdisclosure for use in the treatment of a disease. In an embodiment, thepresent disclosure provides an antigen-binding molecule according to thepresent disclosure or a pharmaceutical composition according to thepresent disclosure for use in the treatment of a disease in anindividual in need thereof. In an embodiment, the present disclosureprovides the use of an antigen-binding molecule according to the presentdisclosure for the manufacture of a medicament. In an embodiment, thepresent disclosure provides the use of an antigen-binding moleculeaccording to the present disclosure for the manufacture of a medicamentfor the treatment of a disease in an individual in need thereof. In anembodiment, the present disclosure pertains to a method of treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a pharmaceutical compositioncomprising an antigen-binding molecule according to the presentdisclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Design of a bivalent or trivalent bispecific antigen-bindingmolecule according to the present disclosure comprising two or three Fvregions.

FIG. 1A: Structure of a bivalent bispecific antigen-binding moleculeaccording to the present disclosure. The structure comprises one Fab arm(Fab₁) from a regular immunoglobulin comprising a first binding siteformed by a first Fv region (Fv₁). A second antigen-binding site isformed by a second Fv region (Fv₂). Each variable domain (VH₂ and VL₂)of the second Fv region (Fv₂) is fused via a peptide linker to theC-terminus of one CH2 domain of an Fc region subunit. The two peptidelinkers may include interchain-cysteines which allows for the formationof stabilizing disulfide bridges between the two linkers (bold crossstrokes). Additional peptide linkers fuse the N-terminus of the VH or VL(VH₂ and VL₂) of the second Fv region (Fv₂) with the CH1 or CL of firstFab. Heterodimerization of the polypeptide chains comprising the two Fcregion subunits is promoted by modification of the CH3 domains in eachFc region subunit by approaches of disulfide stabilization andknob-into-holes technology. In addition, disulfide stabilization in theVH₂/VL₂ interface of the second Fv region may be applied (notindicated). The CH2 domain in each Fc region subunit may be additionallymodified in order to enhance or to abolish Fc mediated effector function(not indicated).

FIG. 1B: Structure of a trivalent bispecific antigen-binding moleculeaccording to the present disclosure comprising a first Fab (Fab₁),second Fab (Fab)₂ and a second Fv region (Fv₂). The molecule comprisestwo Fab arms from a regular immunoglobulin comprising a first and thirdantigen-binding site formed by a first Fv region (Fv₁) and a third Fvregion (Fv₃). A second antigen-binding site is formed by a second Fvregion (Fv₂). Each variable domain (VH₂ and VL₂) of the second Fv region(Fv₂) is fused via a peptide linker to the C-terminus of one CH2 domainof an Fc region subunit. The peptide linkers includeinterchain-cysteines which allow for the formation of stabilizingdisulfide bridges between the two linkers (bold cross strokes).Additional peptide linkers fuse the N-terminus of the VH₂ and VL₂ of thesecond Fv region with the N-terminus of the CH1 or CL of the first Faband second Fab, respectively. Heterodimerization of the polypeptidechains comprising the two Fc region subunits is promoted by modificationof the CH3 domains in each Fc region subunit by approaches of disulfidestabilization and knob-into-holes technology. In addition, disulfidestabilization in the VH₂/VL₂ interface of the second Fv region may beapplied (not indicated). The CH2 domain in each Fc region subunit may beadditionally modified in order to enhance or to abolish Fc mediatedeffector function (not indicated).

FIG. 2: Design of bivalent or trivalent bispecific antigen-bindingmolecules according to the present disclosure comprising two or three Fvregions and additional IgG constant domains.

FIG. 2A: Structure of a bivalent bispecific antigen-binding moleculeaccording to the present disclosure. The molecule comprises one Fab armfrom a regular immunoglobulin comprising a first binding site formed bya first Fv region (Fv₁). A second antigen-binding site is formed by asecond Fv region (Fv₂) of a third Fab (Fab₃). The C-terminus of theheavy and light chain of the third Fab (CH1 or CL, respectively) isfused via peptide linkers to the N-terminus of the CH2 domains of the Fcregion. The peptide linkers include interchain-cysteines, which allowfor the formation of stabilizing disulfide bridges between the twolinkers (bold cross strokes). Additional peptide linkers fuse theN-terminus of the VH or VL of the second Fv region with the C-terminusof the CH1 or CL of the first Fab, respectively. Heterodimerization ofthe polypeptide chains comprising the two Fc region subunits is promotedby modification of the CH3 domains in each Fc region subunit byapproaches of disulfide stabilization and knob-into-holes technology. Inaddition, disulfide stabilization in the VH₂/VL₂ interface of the secondFv region may be applied (not indicated). The CH2 domains may beadditionally modified in order to enhance or to abolish Fc mediatedeffector function (not indicated).

FIG. 2B: Structure of a trivalent bispecific antigen-binding moleculeaccording to the present disclosure comprising a first Fab (Fab₁), asecond Fab (Fab₂) and third Fab (Fab₃). The molecule comprises two Fabarms (Fab₁ and Fab₂) from a regular immunoglobulin comprising a firstand third antigen-binding site formed by a first Fv region (Fv₁) and athird Fv region (Fv₃). A second antigen-binding site is formed by asecond Fv region (Fv₂) of a third Fab (Fab₃). The C-terminus of theconstant domains of the first and third Fab (CH1 or CL, respectively)are fused via peptide linkers to the N-terminus of respective CH2domains of the Fc region. The peptide linkers includeinterchain-cysteines which allow for the formation of stabilizingdisulfide bridges between the two linkers (bold cross strokes).Additional peptide linkers fuse the N-terminus of the VH or VL of thesecond Fv region (Fv₂) with the C-terminus of the CH1 or CL of the firstFab and second Fab, respectively. Heterodimerization of the polypeptidechains comprising the two Fc region subunits is promoted by modificationof the CH3 domains in each Fc region subunit by approaches of disulfidestabilization and knob-into-holes technology. In addition, disulfidestabilization in the VH/VL interface of the second Fv region may beapplied (not indicated). The CH2 domains may be additionally modified inorder to enhance or to inhibit Fc mediated effector function (notindicated).

FIG. 3: Cell binding of 5 mammalian produced and purified bispecifictrivalent antigen-binding molecules according to the present disclosurewith a structure as depicted in FIG. 1B with bivalent binding to HER2and monovalent binding to CD3 (Constructs 1, 3, 4) and negative control(Construct 5). FIG. 3A shows cell binding (signal over background) toCD3 positive Jurkat cells as a function of Construct concentrationdetermined by flow cytometry. FIG. 3B depicts the same as FIG. 3A withthe difference that binding to HER2 positive human adenocarcinoma SKOV-3cells is shown.

FIG. 4: Evaluation of the functional activity of bispecific trivalentantigen-binding molecules according to the present disclosure with astructure as depicted in FIG. 1B with bivalent binding to HER2 andmonovalent binding to CD3 (Constructs 1, 3, 4) and negative control(Construct 5) in a NFAT Reporter Gene Assay using Jurkat cellstransiently transfected with the NFAT reporter gene construct used assurrogate effector cells. As target cells either the HER2 positive humanadenocarcinoma SKOV-3 or the HER2 negative human adenocarcinomaMDA-MB-468 cell line are used. FIG. 4A is a graph showing the relativefluorescence of SKOV-3 cells as a function of Construct concentration.FIG. 4B is a graph showing the relative fluorescence of MDA-MB-468 cellsas a function of Construct concentration.

FIG. 5: Cytotoxicity assay of bispecific trivalent antigen-bindingmolecules according to the present disclosure with a structure asdepicted in FIG. 1B with bivalent binding to HER2 and monovalent bindingto CD3 (Constructs 1, 3, 4) or negative control (Construct 5) on eitherHER2 expressing SKBR3 cells or HER2 negative MDA-MB-468 cells inpresence of human derived PBMCs. Cytotoxic activity of PBMCs is assessedby measuring incorporated CellToxGreen fluorescence. The graph showingthe relative fluorescence of HER2 expressing SKBR3 cells and HERnegative MDA-MV-468 cells as a function of Construct concentration.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure pertains to antigen-binding molecules that aresuited to co-engage two or more antigens simultaneously.

Definitions

The terms “comprising”, “comprises” and “comprised of” “as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,or “composed of”, and are inclusive or open-ended and do not excludeadditional, non-recited members, elements or method steps.

The term “polypeptide” as used herein refer to a polymer of amino acidresidues and does not refer to a specific length of a product. The termapplies to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymers. Unless otherwise indicated, a particularamino acid sequence of a polypeptide also implicitly encompassesconservatively modified variants thereof (e.g. by replacing an aminoacid residue with another amino acid residue having similar structuraland/or chemical properties). A polypeptide may be derived from a naturalbiological source or produced by recombinant technology, but is notnecessarily translated from a designated nucleic acid sequence. It maybe generated in any manner, including chemical synthesis.

The term “antigen-binding molecule” as used herein, refers in itsbroadest sense to a proteinacious molecule that specifically binds to atleast one antigen. An antigen-binding molecule may be composed of one ormore polypeptides. Examples of antigen-binding molecules areimmunoglobulins and derivatives and/or fragments thereof.Antigen-binding molecules according to the present disclosure are basedon a regular immunoglobulin (e.g. IgG) structure that incorporates anadditional Fv region between the two Fab arms and the Fc region. Theantigen-binding molecule as disclosed herein may also lack one of thetwo Fabs arms of a regular IgG. In such an embodiment, the additional Fvregion is incorporated between one Fab arm and the Fc region of aregular immunoglobulin structure. Other proteinaceous antigen-bindingmolecules include scaffolds with antibody-like properties, such asaffibodies (which comprise the Z-domain of protein A), immunity proteins(such as ImmE7), cytochrome b562, proteins comprising ankyrin repeats,PDZ domains or Kunitz domains, insect defensin A, scorpion toxins (suchas charybdotoxin or CTLA-4), knottins (such as Min-23, neocarzinostatin,CBM4-2 or tendamistat), anticalins or armadillo repeat proteins.

The term “antibody” molecule or “immunoglobulin” (Ig) molecule usedherein refers to a protein comprising at least two heavy (H) chains andtwo light (L) chains interconnected by disulfide bonds, which interactswith an antigen. Each heavy chain (HC) is comprised of a heavy chainvariable domain (abbreviated herein as VH) and a heavy chain constantregion. The heavy chain constant region is comprised of three domains,CH1, CH2 and CH3. Each light chain (LC) is comprised of a light chainvariable domain (abbreviated herein as VL) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The VH and VL domains can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FR'sarranged from N-terminus to C-terminus in the following order: FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains of the heavyand light chains (VH and VL) contain a “binding site” or“antigen-binding site” that interacts with an antigen. The constantregions of the antibodies may mediate the binding of the immunoglobulinto host tissues or factors, including various cells of the immune system(e.g., effector cells) and the first component (C1q) of the classicalcomplement system. The term “antibody” includes for example, monoclonalantibodies, human antibodies, humanized antibodies, camelised antibodiesand chimeric antibodies. The antibodies can be of any isotype (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) or subclass. Both the light and heavy chains are dividedinto regions of structural and functional homology.

The term “antibody fragment” as used herein, refers to one or moreportions of an antibody that retain the ability to specifically interactwith (e.g., by binding, steric hindrance, stabilizing spatialdistribution) an antigen. Examples of antibody fragments include, butare not limited to, a Fab, a monovalent fragment consisting of the VL,VH, CL and CH1 domains, wherein the Fab heavy chain (HC) is formed bythe VH and CH1 domains (VH-CH1) and the Fab light chain is formed by thecomplementary VL and CL domains (VL-CL). Accordingly, the Fab heavychain and the Fab light chain are complementary to each other; a F(ab)₂,a bivalent fragment comprising two Fabs linked by a disulfide bridge atthe hinge region; a Fd fragment consisting of the VH and CH1 domains; aFv fragment or Fv region consisting of a dimer of one VL and one VHdomain. Accordingly, the VH and VL domain of a Fv fragment or Fv regionare complementary to each other; a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and an isolatedcomplementarity determining region (CDR). Furthermore, although the twovariable domains of the Fv fragment or Fv region, VL and VH, are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the VL and VH regions pair to form monovalent molecules(referred herein as “single chain Fv” or “scFv”; see e.g., Bird et al.,(1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad.Sci. 85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antibody fragment”. These antibodyfragments are obtained using conventional techniques known to those ofskill in the art, and the fragments are screened for utility in the samemanner as are intact antibodies. Antibody fragments can also beincorporated into single domain antibodies, maxibodies, minibodies,intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv(see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology23:1126-1136). Antibody fragments can be grafted into scaffolds based onpolypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No.6,703,199, which describes fibronectin polypeptide monobodies). Antibodyfragments can be incorporated into single chain molecules comprising apair of tandem Fv segments (VH-CH1-VH-CH1) which, together withcomplementary light chain polypeptides, form a pair of antigen-bindingsites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No.5,641,870).

A “Fv fragment” or “Fv region” is a monovalent antibody fragment, whichconsists of a dimer of one VL and one VH domain. Accordingly, the VH andVL domain of an Fv fragment or Fv region are complementary to eachother.

A “Fab” or “Fab fragment” is a monovalent antibody fragment consistingof the VL, VH, CL and CH1 domains. The Fab heavy chain consists of oneVH and one CH1 domain (VH-CH1) and the Fab light chain consists of oneVL and one CL domain (VL-CL). Accordingly, the Fab heavy chain and theFab light chain are complementary to each other.

The term immunoglobulin (Ig) “hinge” as used herein refers to one of thetwo polypeptides forming the dimeric “hinge region” of animmunoglobulin. The hinge includes the portion of an immunoglobulinheavy chain that joins the CH1 domain to the CH2 domain. Accordingly, anatural occurring immunoglobulin is composed of two identical hinges,which are linked via one or more disulfide bridges formed throughinterchain cysteins present in the two hinges. In other words, a naturaloccurring immunoglobulin is composed of a dimeric disulfide stabilizedhinge region, that joins the two Fab arms of an immunoglobulin to the Fcregion. A hinge can be subdivided into three distinct domains: upper,middle, and lower hinge (Roux et ah, J. Immunol. 1998 161:4083).

The term “Fc region” as used herein refers to the two Fc region subunitsbeing capable of stable association with each other thus forming thedimeric C-terminal region of an immunoglobulin. Accordingly, the two Fcregion subunits ((e.g. the first the second Fc region subunit) arecomplementary to each other. The Fc region of a regular IgG molecule(and of the antigen-binding molecules according to the presentdisclosure) exists as a dimer, each subunit of which comprises the CH2and CH3 IgG heavy chain constant domains. The two subunits of the Fcregion are capable of stable association with each other.

A “Fc region subunit” as used herein refers to one of the twopolypeptides forming the dimeric Fc region of an immunoglobulin or anantigen-binding molecule according to the present disclosure, i.e. apolypeptide comprising C-terminal constant regions of an immunoglobulinheavy chain, capable of stable self-association. Accordingly, the two Fcregion subunits ((e.g. the first the second Fc region subunit) whichform the dimeric Fc region are complementary to each other. For example,IgG Fc region subunit comprises an IgG CH2 and an IgG CH3 constantdomain. The term includes native sequence Fc regions subunits andvariant Fc region subunits. Although the boundaries of the Fc regionsubunits of an IgG heavy chain might vary slightly, the human IgG heavychain Fc region subunit is usually defined to extend from Cys226, orfrom Pro230, to the C-terminus of the heavy chain. However, theC-terminal lysine (Lys447) of the Fc region subunit may or may not bepresent. Unless otherwise specified herein, numbering of amino acidresidues in the Fc region is according to the EU numbering system, alsocalled the EU index, as described in Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991.

A “human antibody” or “human antibody fragment” as used herein, includesantibodies and antibody fragments having variable regions in which boththe framework and CDR regions are derived from sequences of humanorigin. Furthermore, if the antibody contains a constant region, theconstant region also is derived from such sequences. Human originincludes, e.g., human germline sequences, or mutated versions of humangermline sequences or antibody containing consensus framework sequencesderived from human framework sequences analysis, for example, asdescribed in Knappik et al., (2000) J Mol Biol 296:57-86). Thestructures and locations of immunoglobulin variable domains, e.g., CDRs,may be defined using well known numbering schemes, e.g., the Kabatnumbering scheme, the Chothia numbering scheme, or a combination ofKabat and Chothia (see, e.g., Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services (1991), eds.Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabatet al., (1991) Sequences of Proteins of Immunological Interest, 5thedit., NIH Publication no. 91-3242 U.S. Department of Health and HumanServices; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia etal., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol.Biol. 273:927-948. Human antibodies and human variable regions can alsobe isolated from synthetic libraries or from transgenic mice (e.g.xenomouse) provided the respective system yield in antibodies havingvariable regions in which both the framework and CDR regions are derivedfrom sequences of human origin.

The term “chimeric antibody” or “chimeric antibody fragment” is definedherein as an antibody which has constant antibody regions derived from,or corresponding to, sequences found in one species and variableantibody regions derived from another species. Preferably, the constantantibody regions are derived from, or corresponding to, sequences foundin humans, and the variable antibody regions (e.g. VH, VL, CDR or FRregions) are derived from sequences found in a non-human animal, e.g. amouse, rat, rabbit or hamster.

A “humanized antibody” or “humanized antibody fragment” is definedherein as an antibody molecule which has constant antibody regionsderived from sequences of human origin and the variable antibody regionsor parts thereof or only the CDRs are derived from another species.Humanization may be achieved by various methods including, but notlimited to (a) grafting the non-human (e.g., donor antibody) CDRs ontohuman (e.g. recipient antibody) framework and constant regions with orwithout retention of critical framework residues (e.g. those that areimportant for retaining good antigen binding affinity or antibodyfunctions), (b) grafting only the non-human specificity-determiningregions (SDRs or a-CDRs; the residues critical for the antibody-antigeninteraction) onto human framework and constant regions, or (c)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like section by replacement of surface residues. Humanizedantibodies and methods of making them are reviewed, e.g., in Almagro andFransson, Front Biosci 13, 1619-1633 (2008), and are further described,e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al.,Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525(1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984);Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al, Science239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994);Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR)grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing“resurfacing”); Dall'Acqua et al, Methods 36, 43-60 (2005) (describing“FR shuffling”); and Osbourn et al, Methods 36, 61-68 (2005) and Klimkaet al, Br J Cancer 83, 252-260 (2000) (describing the “guided selection”approach to FR shuffling).

The term “isolated” refers to a compound, which can be e.g. an antibody,antibody fragment or antigen-binding molecule, that is substantiallyfree of other antibodies, antibody fragments or antigen-bindingmolecules having different antigenic specificities. Moreover, anisolated antibody, antibody fragment or antigen-binding molecule may besubstantially free of other cellular material and/or chemicals. Thus, insome embodiments, the antibodies, antibody fragments or antigen-bindingmolecules provided are isolated antibodies, antibody fragments orantigen-binding molecules that have been separated from antibodies orantigen-binding molecules with a different specificity. An isolatedantibody or antigen-binding molecule may be a monoclonal antibody,antibody fragment or antigen-binding molecule. An isolated antibody,antibody fragments or antigen-binding molecule may be a recombinantmonoclonal antibody, antibody fragment or antigen-binding molecule. Anisolated antibody, antibody fragment or antigen-binding molecule thatspecifically binds to an epitope, isoform or variant of a target may,however, have cross-reactivity to other related antigens, e.g., fromother species (e.g., species homologs).

The term “recombinant antibody”, “recombinant antibody fragment” or“recombinant antigen-binding molecule”, as used herein, includes allantibodies, antibody fragments or antigen-binding molecules according tothe present disclosure that are prepared, expressed, created orsegregated by means not existing in nature. For example, antibodies orantigen-binding molecules isolated from a host cell transformed toexpress the antibody or antigen-binding molecule, antibodies selectedand isolated from a recombinant, combinatorial human antibody library,and antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of all or a portion of a humanimmunoglobulin gene, sequences to other DNA sequences or antibodiesisolated from an animal (e.g., a mouse) that is transgenic ortranschromosomal for human immunoglobulin genes or a hybridoma preparedtherefrom. Preferably, such recombinant antibodies or antigen-bindingmolecules have variable regions in which the framework and CDR regionsare derived from human germline immunoglobulin sequences. In certainembodiments, however, such recombinant human antibodies can be subjectedto in vitro mutagenesis (or, when an animal transgenic for human Igsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germline VH andVL sequences, may not naturally exist within the human antibody germlinerepertoire in vivo. A recombinant antibody or antigen-binding moleculemay be a recombinant monoclonal antibody or a recombinant monoclonalantigen-binding molecule. In an embodiment, the antibodies and antibodyfragment disclosed herein are isolated from the Ylanthia® antibodylibrary as disclosed in U.S. Ser. No. 13/321,564 or U.S. Ser. No.13/299,367, which both herein are incorporated by reference.

As used herein, the term “monoclonal antibody”, “monoclonal antibodyfragment” or “monoclonal antigen-binding molecule” refers to anantibody, antibody fragment or antigen-binding molecule disclosed hereinthat is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.Monoclonal antibodies or antibody fragments may be made by the hybridomamethod as described in Kohler et al.; Nature, 256:495 (1975) or may beisolated from phage libraries. Other methods for the preparation ofclonal cell lines and monoclonal antibodies or antigen-binding moleculeas disclosed herein expressed thereby are well known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel et al., eds., John Wiley and Sons, New York).

The term “multispecific” means that an antigen-binding molecule is ableto specifically bind to two or more different antigens. Typically, amultispecific antigen-binding molecule comprises of two or moreantigen-binding sites, each of which is specific for a different antigenor epitope. The term “bispecific” means that an antibody orantigen-binding molecule is able to specifically bind to two differentantigens. Typically, a bispecific antigen-binding molecule comprises twoantigen-binding sites, each of which is specific for a different antigenor epitope.

As used herein the term “binds specifically to”, “specifically bindsto”, is “specific to/for” or “specifically recognizes”, or the like,refers to measurable and reproducible interactions such as bindingbetween a target antigen and an antibody, antibody fragment orantigen-binding molecule disclosed herein, which is determinative of thepresence of the target antigen in the presence of a heterogeneouspopulation of molecules including biological molecules. For example, anantibody, antibody fragment or antigen-binding molecule disclosed hereinthat specifically binds to a target antigen (which can be an antigen oran epitope of an antigen) is an antibody, antibody fragment, orantigen-binding molecule that binds this target with greater affinity,avidity, more readily, and/or with greater duration than it binds toother target antigens. In certain embodiments, an antibody, antibodyfragment or antigen-binding molecule specifically binds to an epitope ona protein that is conserved among the protein from different species. Inanother embodiment, specific binding can include, but does not requireexclusive binding. The antibodies, antibody fragments or antigen-bindingmolecules disclosed herein specifically bind to antigens. Methods fordetermining whether two molecules specifically bind are well known inthe art and include, for example, a standard ELISA assay. The scoringmay be carried out by standard color development (e.g. secondaryantibody with horseradish peroxide and tetramethyl benzidine withhydrogen peroxide). The reaction in certain wells is scored by theoptical density, for example, at 450 nm. Typical background (=negativereaction) may be 0.1 OD; typical positive reaction may be 1 OD. Thismeans the difference positive/negative can be more than 5-fold.Typically, determination of binding specificity is performed by usingnot a single reference antigen, but a set of three to five unrelatedantigens, such as milk powder, BSA, transferrin or the like.

As used herein, the term “affinity” refers to the strength ofinteraction between a polypeptide and its target antigen at a singlesite. Within each site, the binding site of the polypeptide interactsthrough weak non-covalent forces with its target at numerous sites; themore interactions, the stronger the affinity.

The term “K_(D)”, as used herein, refers to the dissociation constant,which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and isexpressed as a molar concentration (M). K_(D) values for antigen-bindingmolecules like e.g. monoclonal antibodies or monoclonal antigen-bindingmolecules as disclosed herein can be determined using methods wellestablished in the art. Methods for determining the K_(D) of anantigen-binding molecule like e.g. a monoclonal antibody or monoclonalantigen-binding molecule as disclosed herein are SET (solubleequilibrium titration) or surface plasmon resonance using a biosensorsystem such as a Biacore® system. In the present disclosure an antibodyor antigen-binding molecule according to the present disclosure specificfor an antigen typically has a dissociation rate constant (KD)(koff/kon) of less than 5×10⁻²M, less than 1×10⁻²M, less than 5×10⁻³M,less than 1×10⁻³M, less than 5×10⁻⁴M, less than 1×10⁻⁴M, less than5×10⁻⁵M, less than 1×10⁻⁵M, less than 5×10⁻⁶M, less than 1×10⁻⁶M, lessthan 5×10⁻⁷M, less than 1×10⁻⁷M, less than 5×10⁻⁸M, less than 1×10⁻⁸M,less than 5×10⁻⁹M, less than 1×10⁻⁹M, less than 5×10⁻¹⁰M, less than1×10⁻¹⁰M, less than 5×10⁻¹¹M, less than 1×10⁻¹¹M, less than 5×10⁻¹²M,less than 1×10⁻¹²M, less than 5×10⁻¹³M, less than 1×10⁻¹³M, less than5×10⁻¹⁴M, less than 1×10⁻¹⁴M, less than 5×10⁻¹⁵M, or less than 1×10⁻¹⁵Mor lower for the antigen.

The term “epitope” refers to a site (e.g. a contiguous stretch of aminoacids or a conformational configuration made up of different regions ofnon-contiguous amino acid residues) on a polypeptide or protein, whichis specifically recognized by an antibody, antibody fragment orantigen-binding molecule as disclosed herein, or a T-cell receptor orotherwise interacts with a molecule. Generally, epitopes are ofchemically active surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally may have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. As will be appreciated by one of skill in the art,practically anything to which an antibody or antigen-binding moleculecan specifically bind could be an epitope. An epitope can comprise thoseresidues to which the antibody or antigen-binding molecule binds and maybe “linear” or “conformational.” The term “linear epitope” refers to anepitope wherein all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearly along theprimary amino acid sequence of the protein (continuous). The term“conformational epitope” refers to an epitope in which discontinuousamino acid residues that come together in three dimensionalconformations. In a conformational epitope, the points of interactionoccur across amino acid residues on the protein that are separated fromone another. For example, an epitope can be one or more amino acidresidues within a stretch of amino acid residues as shown by peptidemapping or HDX, or one or more individual amino acid residues as shownby X-ray crystallography.

“Binds the same epitope as” means the ability of an antibody, antibodyfragment or antigen-binding molecule to bind to a specific antigen andbinding to the same epitope as the exemplified antibody orantigen-binding molecule when using the same epitope mapping techniquefor comparing the antibodies or antigen-binding molecules. The epitopesof the exemplified antibody, antigen-binding molecules, other antibodiesand antigen-binding molecules can be determined using epitope mappingtechniques. Epitope mapping techniques are well known in the art. Forexample, conformational epitopes are readily identified by determiningspatial conformation of amino acids such as by, e.g., hydrogen/deuteriumexchange, x-ray crystallography and two-dimensional nuclear magneticresonance.

The terms “engineered” or “modified” as used herein includesmanipulation of nucleic acids or polypeptides by synthetic means (e.g.by recombinant techniques, in vitro peptide synthesis, by enzymatic orchemical coupling of peptides or some combination of these techniques).Preferably, the antibodies, antibody fragments or antigen-bindingmolecules according to the present disclosure are engineered or modifiedto improve one or more properties, such as antigen binding, stability,half-life, effector function, immunogenicity, safety and the like.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen-binding sites in an antigen-binding molecule.

As used herein, the terms “first” and “second” with respect to a Faband/or Fv region, Fc region subunit or the like are used fordistinguishing when there is more than one of each type of component.Use of these terms is not intended to confer a specific order ororientation of the bispecific antigen binding molecule unless explicitlyso stated.

A “modification promoting the association of the first and the second Fcregion subunit” is a manipulation of the polypeptide backbone or thepost-translational modifications of an Fc region subunit that reduces orprevents the association of a polypeptide comprising the Fc regionsubunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc region subunitsdesired to associate (i.e. the first and the second Fc region subunit),wherein the modifications are complementary to each other so as topromote association of the two Fc region subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc region subunits to make their associationsterically or electrostatically favorable, respectively. Accordingly,heterodimerization occurs between a polypeptide comprising the first Fcregion subunit and a polypeptide comprising the second Fc regionsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. Fab, Fv) are not thesame.

As used herein, “amino acid residues” or “amino acid” will be indicatedeither by their full name or according to the standard three-letter orone-letter amino acid code. “Natural occurring amino acids” means thefollowing amino acids:

TABLE 1 Natural occurring amino acids Amino acid Three letter code Oneletter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acidAsp D Cysteine Cys C Glutamic acid Glu E glutamine Gln Q Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made as long as the final construct possesses the desiredcharacteristics, e.g., reduced binding to an Fc receptor, or increasedassociation with another peptide. Amino acid sequence deletions andinsertions include amino- and/or carboxy-terminal deletions andinsertions of amino acid residues. Particular amino acid mutations areamino acid substitutions. Amino acid substitutions include replacementby non-naturally occurring amino acids or by naturally occurring aminoacid derivatives of the twenty standard amino acids. Amino acidmutations can be generated using genetic or chemical methods well knownin the art. Genetic methods may include site-directed mutagenesis, PCR,gene synthesis and the like. It is contemplated that methods of alteringthe side chain group of an amino acid residue by methods other thangenetic engineering, such as chemical modification, may also be useful.Various designations may be used herein to indicate the same amino acidmutation. For example, a substitution from glyince at position 327 ofthe Fc region to alanine can be indicated as 237A, G337, G337A, orGly329Ala.

The term “vector” refers to a polynucleotide molecule capable oftransporting another polynucleotide to which it has been linked.Preferred vectors are those capable of autonomous replication and/orexpression of nucleic acids to which they are linked. One type of vectoris a “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments may be ligated. Another type of vector isa viral vector, wherein additional DNA segments may be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and mammalian vectors). Other vectorscan be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Vectors may be compatible with prokaryotic or eukaryotic cells.Prokaryotic vectors typically include a prokaryotic replicon, which mayinclude a prokaryotic promoter capable of directing the expression(transcription and translation) of the peptide in a bacterial host cell,such as Escherichia coli transformed therewith. A promoter is anexpression control element formed by a DNA sequence that permits bindingof RNA polymerase and transcription to occur. Promoter sequencescompatible with bacterial hosts are typically provided in plasmidvectors containing convenience restriction sites for insertion of a DNAsegment. Examples of such vector plasmids include pUC8, pUC9, pBR322,and pBR329, pPL and pKK223, available commercially. “Expression vectors”are those vectors capable of directing the expression of nucleic acidsto which they are operatively linked and is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions. The expression vectorincludes an expression cassette into which the nucleic acid sequenceencoding an antigen-binding molecule according to the present disclosure(i.e. the coding region) is cloned in operable association with apromoter and/or other transcription or translation control elements. Asused herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, if present, but any flankingsequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5′ and 3′ untranslated regions,and the like, are not part of a coding region.

As used herein, the term “host cell” refers to any kind of cellularsystem which can be engineered to generate an antigen-binding moleculeaccording to the present disclosure and refers to a cell into which a(recombinant expression) vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. Typical host cells are prokaryotic(such as bacterial, including but not limited to E. coli) or eukaryotic(which includes yeast, mammalian cells, and more). Bacterial cells arepreferred prokaryotic host cells and typically are a strain ofEscherichia coli (E. coli) such as, for example, the E. coli strain DH5available from Bethesda Research Laboratories, Inc., Bethesda, Md.Preferred eukaryotic host cells include yeast and mammalian cellsincluding murine and rodents, preferably vertebrate cells such as thosefrom a mouse, rat, monkey or human cell line, for example HKB11 cells,PERC.6 cells, or CHO cells.

The term “EC₅₀” as used herein, refers to the concentration of anantibody, antibody fragment or antigen-binding molecule as disclosedherein, which induces a response in an assay half way between thebaseline and maximum. It therefore represents the antibody orantigen-binding molecule concentration at which 50% of the maximaleffect is observed.

The terms “inhibition” or “inhibit” or “reduction” or “reduce” or“neutralization” or “neutralize” refer to a decrease or cessation of anyphenotypic characteristic (such as binding, a biological activity orfunction) or to the decrease or cessation in the incidence, degree, orlikelihood of that characteristic. The “inhibition”, “reduction” or“neutralization” needs not to be complete as long as it is detectableusing an appropriate assay. In some embodiments, by “reduce” or“inhibit” is meant the ability to cause a decrease of 20% or greater. Inanother embodiment, by “reduce” or “inhibit” is meant the ability tocause a decrease of 50% or greater. In yet another embodiment, by“reduce” or “inhibit” is meant the ability to cause an overall decreaseof 75%, 85%, 90%, 95%, or greater.

The terms “increase” or “enhance” refer to an increase of any phenotypiccharacteristic (such as binding, a biological activity or function) orto the increase in the incidence, degree, or likelihood of thatcharacteristic. The “increase” or “enhance” needs not to be maximumeffect as long as it is detectable using an appropriate assay. In someembodiments, by “increase” or “enhance” is meant the ability to cause anincrease of 20% or greater. In another embodiment, by “increase” or“enhance” is meant the ability to cause an increase of 50% or greater.In yet another embodiment, by “increase” or “enhance” is meant theability to cause an overall increase of 75%, 85%, 90%, 95%, or greater.

The term “antagonistic” antigen-binding molecule as used herein refersto an antigen-binding molecule that interacts with an antigen andpartially or fully inhibits or neutralizes a biological activity orfunction or any other phenotypic characteristic of an target antigen.

The term “agonistic” antigen-binding molecule as used herein refers toan antigen-binding molecule that interacts with an antigen and increasesor enhances a biological activity or function or any other phenotypiccharacteristic of the target antigen.

An “effective amount” of an agent, e.g. a pharmaceutical composition,refers to the amount that is necessary to result in a physiologicalchange in the cell or tissue to which it is administered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

The terms “individual” or “subject” refer to a mammal.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

The term “pharmaceutically acceptable carrier” refers to an ingredientin a pharmaceutical composition, other than an active ingredient, whichis nontoxic to a subject. A pharmaceutically acceptable carrierincludes, but is not limited to, a buffer, excipient, stabilizer, orpreservative.

As used herein, “treatment”, “treat” or “treating” and the like refersto clinical intervention in an attempt to alter the natural course of adisease in the individual being treated, and can be performed either forprophylaxis or during the course of clinical pathology. Desirableeffects of treatment include, but are not limited to, preventingoccurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antigen-bindingmolecules according to the preset disclosure are used to delaydevelopment of a disease or to slow the progression of a disease.

The term “effector function” refers to those biological activitiesattributable to the Fc region of an antibody or antigen-bindingmolecules according to the present disclosure, which vary with theantibody isotype. Examples of antibody effector functions include C1qbinding and complement dependent cytotoxicity (CDC); Fc receptor bindingand antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;down regulation of cell surface receptors (e.g. B cell receptor); and Bcell activation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which antibodies or antigen-binding moleculesaccording to the present disclosure bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. NK cells, neutrophils, andmacrophages) enable these cytotoxic effector cells to bind specificallyto an antigen-bearing target cell and subsequently kill the target cellwith cytotoxins. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)or to antigen-binding molecules of the present disclosure, which arebound to their cognate antigen. To assess complement activation, a CDCassay, e.g., as described in Gazzano-Santoro et al, J. Immunol. Methods202:163 (1996), may be performed. Polypeptide variants with altered Fcregion subunit amino acid sequences (polypeptides with a variant ormodified Fc region subunit) and increased or decreased C1q bindingcapability are described, e.g., in U.S. Pat. No. 6,194,551 and WO1999/51642. See also, e.g., Idusogie et al. J. Immunol. 164: 4178-4184(2000).

Embodiments

The present disclosure pertains to antigen-binding molecules that aresuited to co-engage two or more (different) antigens simultaneously. Theability to target two or more different antigens with different valency(e.g. one antigen monovalently and one antigen bivalently) is aparticular useful aspect of the antigen-binding molecules disclosedherein.

The individual components of an antigen-binding molecule according tothe present disclosure can be used to each other in a variety ofconfigurations. Exemplary configurations are depicted in FIGS. 1 and 2.

In general, there are two main types of antigen-binding molecules asdescribed herein:

-   -   a. one type that utilizes two Fv regions. This type allows        for (I) simultaneous monovalent binding to two (different)        antigens or (II) bivalent binding to one antigen.    -   b. one type that utilizes three Fv regions. This type allows        for (I) simultaneous bivalent binding to one antigen and        monovalent binding to second antigen or (II) trivalent binding        to one antigen.

Multispecific Antigen-Binding Molecules

An antigen-binding molecule according to the present disclosure can bemade of two or more, preferably three Fv regions. Accordingly, thepresent molecule can act as a bivalent or trivalent antigen-bindingmolecule. The basic structures of antigen-binding molecules according tothe present disclosure are depicted in FIGS. 1 and 2.

Bivalent Antigen-Binding Molecules

In an embodiment, an antigen-binding molecule according to the presentdisclosure is composed of two Fv regions. This is achieved by using aregular immunoglobulin (e.g. IgG) antibody structure (which lacks one ofthe two Fab arms) that incorporates an additional Fv region between theretained Fab arm and the Fc portion of the immunoglobulin structure.

In an embodiment, an antigen-binding molecule according to the presentdisclosure allows for a monovalent binding to two different antigens. Insuch an embodiment, the antigen-binding molecule comprises at least twoFv regions, wherein one Fv region binds to a first antigen and the otherFv region binds to a second antigen. In such an embodiment, theantigen-binding molecule according to the present disclosure refers to abivalent bispecific antigen-binding molecule.

In an embodiment, the present disclosure pertain to an antigen-bindingmolecule, comprising

-   -   a) a first Fab comprising a first Fv region, which specifically        binds to a first antigen,    -   b) a second Fv region which specifically binds to a second        antigen and    -   c) a Fc region composed of a first and second Fc region subunit;        wherein        -   I. the C-terminus of the heavy or light chain of the first            Fab is fused to the N-terminus of the VH or VL of the second            Fv region, and wherein        -   II. the C-terminus of the VH or VL of the second Fv region            is fused to the N-terminus of the first Fc region subunit            and the N-terminus of the second Fc domain subunit is fused            to the C-terminus of the complementary variable domain of            the second Fv region.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region. In anembodiment, the C-terminus of the CH1 of the first Fab is fused to theN-terminus of the VH of the second Fv region. In an embodiment, thefusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region. In anembodiment, the C-terminus of the CH1 of the first Fab is fused to theN-terminus of the VL of the second Fv region. In an embodiment, thefusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region. In anembodiment, the C-terminus of the CL of the first Fab is fused to theN-terminus of the VH of the second Fv region.

In an embodiment, the fusion occurs via a peptide linker. In anembodiment, the C-terminus of the light chain of the first Fab is fusedto the N-terminus of the VL of the second Fv region.

In an embodiment, the C-terminus of the CL of the first Fab is fused tothe N-terminus of the VL of the second Fv region. In an embodiment, thefusion occurs via a peptide linker.

In an embodiment, the C-terminus of the VH of the second Fv region isfused to the N-terminus of the first Fc region subunit. In anembodiment, the fusion occurs via a peptide linker. In an embodiment,the C-terminus of the VH of the second Fv region is fused to theN-terminus of the second Fc region subunit. In an embodiment, the fusionoccurs via a peptide linker. In an embodiment, the C-terminus of the VLof the second Fv region is fused to the N-terminus of the first Fcregion subunit. In an embodiment, the fusion occurs via a peptidelinker. In an embodiment, the C-terminus of the VL of the second Fvregion is fused to the N-terminus of the second Fc region subunit. In anembodiment, the fusion occurs via a peptide linker. In an embodiment,the N-terminus of the first Fc domain subunit is fused to the C-terminusof the VH of the second Fv region. In an embodiment, the fusion occursvia a peptide linker. In an embodiment, the N-terminus of the first Fcdomain subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the fusion occurs via a peptide linker.

In an embodiment, the N-terminus of the second Fc domain subunit isfused to the C-terminus of the VH of the second Fv region. In anembodiment, the fusion occurs via a peptide linker. In an embodiment,the N-terminus of the second Fc domain subunit is fused to theC-terminus of the VL of the second Fv region. In an embodiment, thefusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit. In an embodiment, the C-terminus of theCH1 of the first Fab is fused to the N-terminus of the VH of the secondFv region and the C-terminus of the VH of the second Fv region is fusedto the N-terminus of the first Fc region subunit. In an embodiment, eachfusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit. In an embodiment, the C-terminus of theCH1 of the first Fab is fused to the N-terminus of the VH of the secondFv region and the C-terminus of the VH of the second Fv region is fusedto the N-terminus of the second Fc region subunit. In an embodiment,each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit. In an embodiment, the C-terminus of theCH1 of the first Fab is fused to the N-terminus of the VH of the secondFv region and the C-terminus of the VL of the second Fv region is fusedto the N-terminus of the first Fc region subunit. In an embodiment, eachfusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit. In an embodiment, the C-terminus of theCH1 of the first Fab is fused to the N-terminus of the VH of the secondFv region and the C-terminus of the VL of the second Fv region is fusedto the N-terminus of the second Fc region subunit. In an embodiment,each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit. In an embodiment, the C-terminus of theCH1 of the first Fab is fused to the N-terminus of the VL of the secondFv region and the C-terminus of the VH of the second Fv region is fusedto the N-terminus of the first Fc region subunit. In an embodiment, eachfusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit. In an embodiment, the C-terminus of theCH1 of the first Fab is fused to the N-terminus of the VL of the secondFv region and the C-terminus of the VH of the second Fv region is fusedto the N-terminus of the second Fc region subunit. In an embodiment,each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit. In an embodiment, the C-terminus of theCH1 of the first Fab is fused to the N-terminus of the VL of the secondFv region and the C-terminus of the VL of the second Fv region is fusedto the N-terminus of the first Fc region subunit. In an embodiment, eachfusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit. In an embodiment, the C-terminus of theCH1 of the first Fab is fused to the N-terminus of the VL of the secondFv region and the C-terminus of the VL of the second Fv region is fusedto the N-terminus of the second Fc region subunit. In an embodiment,each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit. In an embodiment, the C-terminus of theCL of the first Fab is fused to the N-terminus of the VH of the secondFv region and the C-terminus of the VH of the second Fv region is fusedto the N-terminus of the first Fc region subunit. In an embodiment, eachfusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit. In an embodiment, the C-terminus of theCL of the first Fab is fused to the N-terminus of the VH of the secondFv region and the C-terminus of the VH of the second Fv region is fusedto the N-terminus of the second Fc region subunit. In an embodiment,each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit. In an embodiment, the C-terminus of theCL of the first Fab is fused to the N-terminus of the VH of the secondFv region and the C-terminus of the VL of the second Fv region is fusedto the N-terminus of the first Fc region subunit. In an embodiment, eachfusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit. In an embodiment, the C-terminus of theCL of the first Fab is fused to the N-terminus of the VH of the secondFv region and the C-terminus of the VL of the second Fv region is fusedto the N-terminus of the second Fc region subunit. In an embodiment,each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit. In an embodiment, the C-terminus of theCL of the first Fab is fused to the N-terminus of the VL of the secondFv region and the C-terminus of the VH of the second Fv region is fusedto the N-terminus of the first Fc region subunit. In an embodiment, eachfusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit. In an embodiment, the C-terminus of theCL of the first Fab is fused to the N-terminus of the VL of the secondFv region and the C-terminus of the VH of the second Fv region is fusedto the N-terminus of the second Fc region subunit. In an embodiment,each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit. In an embodiment, the C-terminus of theCL of the first Fab is fused to the N-terminus of the VL of the secondFv region and the C-terminus of the VL of the second Fv region is fusedto the N-terminus of the first Fc region subunit. In an embodiment, eachfusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit. In an embodiment, the C-terminus of theCL of the first Fab is fused to the N-terminus of the VL of the secondFv region and the C-terminus of the VL of the second Fv region is fusedto the N-terminus of the second Fc region subunit. In an embodiment,each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the C-terminus of the CH1 of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the C-terminus of the CH1 of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcdomain subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, the C-terminus of the CH1 of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcdomain subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcdomain subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, the C-terminus of the CH1 of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcdomain subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the C-terminus of the CH1 of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the C-terminus of the CH1 of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, the C-terminus of the CH1 of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, the C-terminus of the CH1 of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the C-terminus of the CL of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the C-terminus of the CL of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, the C-terminus of the CL of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, the C-terminus of the CL of the first Fab isfused to the N-terminus of the VH of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the C-terminus of the CL of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, the C-terminus of the CL of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VH of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VL of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, the C-terminus of the CL of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the first Fc region subunit and the N-terminus of the second Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, the C-terminus of the CL of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the VL of the second Fv region is fused to the N-terminusof the second Fc region subunit and the N-terminus of the first Fcregion subunit is fused to the C-terminus of the VH of the second Fvregion. In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, an antigen-binding molecule according to the presentdisclosure is composed of at least 3 polypeptides, wherein

-   -   a. a first polypeptide comprises the light or heavy chain of the        first Fab,    -   b. a second polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary light or heavy chain of the first Fab,        -   ii. the VH or VL of the second Fv region and        -   iii. the first or second Fc region subunit    -   c. a third polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary VH or VL of the second Fv region and        -   ii. the complementary first or second Fc region subunit.

In an embodiment, the first polypeptide comprises the light chain of thefirst Fab. In an embodiment, the first polypeptide comprises the heavychain of the first Fab.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary heavy chain of the first Fab,    -   ii. the VH of the second Fv region and    -   iii. the first Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary heavy chain of the first Fab,    -   ii. the VL of the second Fv region and    -   iii. the first Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary heavy chain of the first Fab,    -   ii. the VH of the second Fv region and    -   iii. the second Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary heavy chain of the first Fab,    -   ii. the VL of the second Fv region and    -   iii. the second Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary light chain of the first Fab,    -   ii. the VH of the second Fv region and    -   iii. the first Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary light chain of the first Fab,    -   ii. the VL of the second Fv region and    -   iii. the first Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary light chain of the first Fab,    -   ii. the VH of the second Fv region and    -   iii. the second Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary light chain of the first Fab,    -   ii. the VL of the second Fv region and    -   iii. the second Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the complementary VH of the second Fv region and    -   ii. the complementary first Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the complementary VH of the second Fv region and    -   ii. the complementary second Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the complementary VL of the second Fv region and    -   ii. the complementary first Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the complementary VL of the second Fv region and    -   ii. the complementary second Fc region subunit.

In an embodiment, an antigen-binding molecule according to the presentdisclosure is composed of at least 3 polypeptides, wherein

-   -   a. a first polypeptide comprises the light chain of the first        Fab,    -   b. a second polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary heavy chain of the first Fab,        -   ii. the VL of the second Fv region and        -   iii. the first Fc region subunit    -   c. a third polypeptide comprises from its N-terminus to its        C-terminus    -   i. the complementary VH of the second Fv region and    -   ii. the complementary second Fc region subunit.

In an embodiment, an antigen-binding molecule according to the presentdisclosure is composed of at least 3 polypeptides, wherein

-   -   a. a first polypeptide comprises the light chain of the first        Fab,    -   b. a second polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary heavy chain of the first Fab,        -   ii. the VH of the second Fv region and        -   iii. the first Fc region subunit    -   c. a third polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary VL of the second Fv region and        -   ii. the complementary second Fc region subunit.

In an embodiment, an antigen-binding molecule according to the presentdisclosure is composed of 3 polypeptides.

According to the aforementioned embodiments, the first Fv region and thesecond Fv region can be of different antigen specificity and are fusedto each other in a configuration, which allows for a geometry anddistance between the two Fv regions different from that of thebispecific antibody formats known in the art.

An antigen-binding molecule according to the present disclosure providesmonovalent binding to at least one of the antigens it binds to.Monovalent binding may be desired or required in situations whereinternalization of the target antigen occurs following bivalent bindingof an antigen-binding molecule. In such cases, bivalent binding to onetarget antigen may enhance internalization of the antigen, therebyreducing its availability. Furthermore, monovalent binding is essentialwhere crosslinking of a target antigen is not desired. For example,bivalent binding to certain target classes, such as receptor tyrosinekinase, may mimic the function of natural ligands resulting in receptoractivation rather an inactivation.

The configuration present in an antigen-binding molecule according tothe present disclosure is particularly suited to mimic the immunologicalsynapse between a T-cell and a target cell, as required, if a bispecificantigen-binding molecule is to be used for T-cell engagement andredirection. Ensuring monovalent binding to an activating T-cell antigen(such as CD3) minimizes the risk of activation of said T-cell in theabsence of target cells.

However, bivalent binding to a target antigen might be also desirable incertain situations, for example to increase binding affinity and tooptimize targeting.

Trivalent Antigen-Binding Molecules

In an preferred embodiment, an antigen-binding molecule according to thepresent disclosure is composed of three Fv regions. This is achieved byusing a regular immunoglobulin (e.g. IgG) antibody structure (two heavychains with associated two light chains that form two Fv regions) thatincorporates an additional Fv region between the two Fab arms and the Fcportion of the regular immunoglobulin structure.

In an embodiment, an antigen-binding molecule according to the presentdisclosure comprises a second Fab comprising a third Fv region, whichspecifically binds to a third antigen.

In an embodiment, the third antigen is identical to the first or secondantigen. In an embodiment, the third antigen is identical to the firstantigen. In an embodiment, the third antigen is identical to the secondantigen.

In an embodiment, an antigen-binding molecule according to the presentdisclosure comprises a second Fab comprising a third Fv region, whichspecifically binds to the first or the second antigen.

In an embodiment, an antigen-binding molecule according to the presentdisclosure comprises a first Fab comprising a first Fv region, whichspecifically binds to a first antigen, a second Fab comprising a thirdFv region, which specifically binds to a third antigen and a second Fvregion, which specifically binds to a second antigen. In an embodiment,the third antigen is identical to the first or second antigen. In anembodiment, the third antigen is identical to the first antigen.

In an embodiment, the second Fab is fused to the second Fv region. In anembodiment, the C-terminus of the second Fab is fused to the N-terminusof the second Fv region. In an embodiment, the second Fab is fused tothe second Fv region via a peptide linker.

In an embodiment, the present disclosure provides an antigen-bindingmolecule, comprising

-   -   a) a first Fab comprising a first Fv region, which specifically        binds to a first antigen,    -   b) a second Fv region which specifically binds to a second        antigen and    -   c) a second Fab comprising a third Fv region, which specifically        binds to a third antigen, and    -   d) a Fc region composed of a first and second Fc region subunit;        wherein        -   I. the C-terminus of the heavy or light chain of the first            Fab is fused to the N-terminus of the VH or VL of the second            Fv region, and wherein            -   II. the C-terminus of the VH or VL of the second Fv                region is fused to the N-terminus of the first Fc region                subunit and the N-terminus of the second Fc domain                subunit is fused to the C-terminus of the complementary                variable domain of the second Fv region, and wherein            -   III. the C-terminus of the heavy or light chain of the                second Fab is fused to the N-terminus of the VH or VL of                the second Fv region with the proviso that the first and                second Fab are fused to distinct variable domains of the                second Fv region.

In an embodiment, each fusion occurs via a peptide linker.

In an embodiment, the antigen-binding molecule according to the presentdisclosure comprises not more than three Fv domains. In an embodiment,the antigen-binding molecule according to the present disclosureconsists of three Fv domains.

In an embodiment, the C-terminus of the CH1 or CL of the second Fab isfused to the N-terminus of the VH or VL of the second Fv region with theproviso that the first and second Fab are fused to distinct variabledomains of the second Fv region.

In an embodiment, the C-terminus of the heavy chain of the second Fab isfused to the N-terminus of the VH of the second Fv region with theproviso that first and second Fab are fused to distinct variable domainsof the second Fv region. In an embodiment, the C-terminus of the CH1 ofthe second Fab is fused to the N-terminus of the VH of the second Fvregion with the proviso that first and second Fab are fused to distinctvariable domains of the second Fv region. In an embodiment, the fusionoccurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the second Fab isfused to the N-terminus of the VL of the second Fv region with theproviso that first and second Fab are fused to distinct variable domainsof the second Fv region. In an embodiment, the C-terminus of the CH1 ofthe second Fab is fused to the N-terminus of the VL of the second Fvregion with the proviso that first and second Fab are fused to distinctvariable domains of the second Fv region. In an embodiment, the fusionoccurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the second Fab isfused to the N-terminus of the VH of the second Fv region with theproviso that first and second Fab are fused to distinct variable domainsof the second Fv region. In an embodiment, the C-terminus of the CL ofthe second Fab is fused to the N-terminus of the VH of the second Fvregion with the proviso that first and second Fab are fused to distinctvariable domains of the second Fv region. In an embodiment, the fusionoccurs via a peptide linker.

In an embodiment, the C-terminus of the light chain of the second Fab isfused to the N-terminus of the VL of the second Fv region with theproviso that first and second Fab are fused to distinct variable domainsof the second Fv region. In an embodiment, the C-terminus of the CL ofthe second Fab is fused to the N-terminus of the VL of the second Fvregion with the proviso that first and second Fab are fused to distinctvariable domains of the second Fv region. In an embodiment, the fusionoccurs via a peptide linker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the heavy chain of the second Fab is fused to theN-terminus of the VH of the second Fv region. In an embodiment, theC-terminus of the heavy chain of the first Fab is fused to theN-terminus of the VH of the second Fv region and the C-terminus of theheavy chain of the second Fab is fused to the N-terminus of the VL ofthe second Fv region.

In an embodiment, the C-terminus of the light chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the light chain of the second Fab is fused to theN-terminus of the VH of the second Fv region. In an embodiment, theC-terminus of the light chain of the first Fab is fused to theN-terminus of the VL of the second Fv region and the C-terminus of thelight chain of the second Fab is fused to the N-terminus of the VL ofthe second Fv region. In an embodiment, the fusion occurs via a peptidelinker.

In an embodiment, the C-terminus of the heavy chain of the first Fab isfused to the N-terminus of the VL of the second Fv region and theC-terminus of the light chain of the second Fab is fused to theN-terminus of the VH of the second Fv region. In an embodiment, theC-terminus of the heavy chain of the first Fab is fused to theN-terminus of the VH of the second Fv region and the C-terminus of thelight chain of the second Fab is fused to the N-terminus of the VL ofthe second Fv region. In an embodiment, the C-terminus of the lightchain of the first Fab is fused to the N-terminus of the VL of thesecond Fv region and the C-terminus of the heavy chain of the second Fabis fused to the N-terminus of the VH of the second Fv region. In anembodiment, the C-terminus of the light chain of the first Fab is fusedto the N-terminus of the VH of the second Fv region and the C-terminusof the heavy chain of the second Fab is fused to the N-terminus of theVL of the second Fv region. In an embodiment, the fusion occurs via apeptide linker.

In an embodiment, the antigen-binding molecule according to the presentdisclosure is composed of at least 4 polypeptides, wherein

-   -   a. a first polypeptide comprises the light or heavy chain of the        first Fab,    -   b. a second polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary light or heavy chain of the first Fab,        -   ii. the VH or VL of the second Fv region and        -   iii. the first or second Fc domain subunit    -   c. a third polypeptide comprises from its N-terminus to its        C-terminus        -   i. the light or heavy chain of the second Fab,        -   ii. the complementary VH or VL of the second Fv region and        -   iii. the complementary first or second Fc domain subunit    -   d. a fourth polypeptide comprises the complementary light or        heavy chain of the second Fab.

In an embodiment, the first polypeptide comprises the light chain of thefirst Fab. In an embodiment, the light chain of the first Fab comprisesthe VL and CL of the first Fab. In an embodiment, the first polypeptidecomprises the heavy chain of the first Fab. In an embodiment, the heavychain of the first Fab comprises the VH and CH1 of the first Fab.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary heavy chain of the first Fab,    -   ii. the VH of the second Fv region and    -   iii. the first Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary heavy chain of the first Fab,    -   ii. the VL of the second Fv region and    -   iii. the first Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary heavy chain of the first Fab,    -   ii. the VH of the second Fv region and    -   iii. the second Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary heavy chain of the first Fab,    -   ii. the VL of the second Fv region and    -   iii. the second Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary light chain of the first Fab,    -   ii. the VH of the second Fv region and    -   iii. the first Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary light chain of the first Fab,    -   ii. the VL of the second Fv region and    -   iii. the first Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary light chain of the first Fab,    -   ii. the VH of the second Fv region and    -   iii. the second Fc region subunit.

In an embodiment, the second polypeptide comprises from its N-terminusto its C-terminus

-   -   i. the complementary light chain of the first Fab,    -   ii. the VL of the second Fv region and    -   iii. the second Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the heavy chain of the second Fab,    -   ii. the complementary VH of the second Fv region and    -   iii. the complementary first Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the heavy chain of the second Fab,    -   ii. the complementary VH of the second Fv region and    -   iii. the complementary second Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the heavy chain of the second Fab,    -   ii. the complementary VL of the second Fv region and    -   iii. the complementary first Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the heavy chain of the second Fab,    -   ii. the complementary VL of the second Fv region and    -   iii. the complementary second Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the light chain of the second Fab,    -   ii. the complementary VH of the second Fv region and    -   iii. the complementary first Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the light chain of the second Fab,    -   ii. the complementary VH of the second Fv region and    -   iii. the complementary second Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the light chain of the second Fab,    -   ii. the complementary VL of the second Fv region and    -   iii. the complementary first Fc region subunit.

In an embodiment, the third polypeptide comprises from its N-terminus toits C-terminus

-   -   i. the light chain of the second Fab,    -   ii. the complementary VL of the second Fv region and    -   iii. the complementary second Fc region subunit.

In an embodiment, the fourth polypeptide comprises the complementarylight chain of the second Fab. In an embodiment, the fourth polypeptidecomprises the complementary heavy chain of the second Fab. In anembodiment, an antigen-binding molecule according to the presentdisclosure is composed of 4 polypeptides. In an embodiment, anantigen-binding molecule according to the present disclosure, iscomposed of at least 4 polypeptides, wherein

-   -   a. a first polypeptide comprises the light chain of the first        Fab,    -   b. a second polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary heavy chain of the first Fab,        -   ii. the VL of the second Fv region and        -   iii. the first Fc domain subunit    -   c. a third polypeptide comprises from its N-terminus to its        C-terminus        -   i. the heavy chain of the second Fab,        -   ii. the complementary VH of the second Fv region and        -   iii. the complementary second Fc domain subunit    -   d. a fourth polypeptide comprises the complementary light chain        of the second Fab.

In an embodiment, an antigen-binding molecule according to the presentdisclosure, is composed of at least 4 polypeptides, wherein

-   -   a. a first polypeptide comprises the light chain of the first        Fab,    -   b. a second polypeptide comprises from its N-terminus to its        C-terminus        -   i. the complementary heavy chain of the first Fab,        -   ii. the VH of the second Fv region and        -   iii. the first Fc domain subunit    -   c. a third polypeptide comprises from its N-terminus to its        C-terminus        -   i. the heavy chain of the second Fab,        -   ii. the complementary VL of the second Fv region and        -   iii. the complementary second Fc domain subunit    -   d. a fourth polypeptide comprises the complementary light chain        of the second Fab.

In an embodiment, the light chain of the first or second Fab comprisesthe VL and CL of the first or second Fab, respectively. In anembodiment, the light chain of the first or second Fab consists of theVL and CL of the first or second Fab, respectively.

In an embodiment, the third antigen is identical to the first antigen.

In an embodiment, the antigen-binding molecule according to the presentdisclosure provides bivalent binding to the first antigen and monovalentbinding to the second antigen.

In an embodiment, the antigen-binding molecule according to the presentdisclosure is a trivalent bispecific antigen-binding molecule.

In an embodiment, the first antigen is a tumor-associated antigen. In anembodiment, the first antigen is a tumor-associated antigen expressed ona tumor cell.

In an embodiment, the second antigen is an immune cell related antigen.In an embodiment, the second antigen is expressed on an immune cell. Inan embodiment, the second antigen is expressed on an immune effectorcell. In an embodiment, the second antigen is expressed on a cytotoxicT-cell. In an embodiment, the second antigen is CD3.

In an embodiment, the Fc region is an IgG1 Fc region. In an embodiment,said IgG1 Fc region is a human IgG1 Fc region. In an embodiment, the Fcregion comprises one or more amino acid modifications promoting theassociation of the first and second Fc region subunit.

In an embodiment, in the CH3 domain of first Fc region subunit, thethreonine residue at position 366 is replaced with a tryptophan residue(T366W) and the serine residue at position 354 is replaced with acysteine residue (S354C) and in the CH3 domain of the second Fc regionsubunit the tyrosine residue at position 407 is replaced with a valineresidue (Y407V), the threonine residue at position 366 is replaced witha serine residue (T366S), the leucine residue at position 368 isreplaced with an alanine residue (L368A) and the tyrosine residue atposition 349 is replaced by a cysteine residue (Y349C) with numberingaccording EU index.

In an embodiment, in each Fc region subunit, at least 5 amino acidresidues in the positions corresponding to positions L234, L235, G237,A330, P331 with numbering according EU index in a human IgG1 are mutatedto A, E, A, S, and S, respectively.

Antibodies

The antibodies or antibody fragments, or heavy and light chain variableregions used in an antigen-binding molecule according to the presentdisclosure can be of any animal species origin, such as murine, rat,human or non-human primate. Preferably, the origin is human or may bealso obtained by humanization approaches.

Accordingly, the Fab and/or Fv regions used in the antigen-bindingmolecules according to the present disclosure are human or humanized. Inan embodiment, the Fv region is human. In an embodiment, the Fv regionis humanized. In an embodiment, the Fab is human. In an embodiment, theFab is humanized. In yet another embodiment, the Fab comprises humanheavy and light chain constant regions.

Linkers

An antigen-binding molecule according to the present disclosure can bedesigned such that its individual components (e.g. Fab, Fv region, Fcregion, variable domains, constant domains) are fused directly to eachother or indirectly through a linker.

In certain embodiments, the individual components of an antigen-bindingmolecule according to the present disclosure are genetically fused toeach other. Such fusion can be achieved by a number of strategies, whichinclude, but are not limited to polypeptide fusion between the N- andC-terminus of polypeptides, fusion via disulfide bonds, and fusion viachemical cross-linking reagents.

A variety of linkers may be used in the embodiments described herein tocovalently fuse the individual components of an antigen-binding moleculeaccording to the present disclosure to its intended fusion partner. Thecomposition and length of a linker may be determined in accordance withmethods well known in the art and may be tested for efficacy.Preferably, the linker is non-immunogenic. In an embodiment, the linkeris a peptide linker. In an embodiment, the linker is a peptide linkercomprising one or more amino acid residues, joined by peptide bonds thatare known in the art. The peptide linker should have a length that isadequate to fuse two polypeptides (or components) in such a way thatthey assume the correct conformation relative to one another so thatthey retain or obtain the desired activity.

In an embodiment, a peptide linker according to the present disclosureis from 1 to 70 amino residues in length, 1 to 50 amino acid residues inlength, 1 to 40 amino residues in length, 1 to 30 amino acid residues inlength, 1 to 20 amino acid residues in length, 1 to 10 amino acidresidue in length, 1 to 5 amino acid residues in length.

In an embodiment, a peptide linker according to the present disclosurehas a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70 amino acidsresidues. In an embodiment, a peptide linkers linker according to thepresent disclosure has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, or 70amino acids residues

The peptide linker may pre-dominantly comprise the following amino acidresidues: Gly, Ser, Ala, or Thr. Suitable, non-immunogenic peptidelinkers comprises glycine-serine polymers for example, (GS)_(n) (SEQ IDNO: 34), (G₄S)_(n) (SEQ ID NO: 35), (SG₄)_(n) (SEQ ID NO: 36),(GSGGS)_(n) (SEQ ID NO: 37), (GGGS)_(n) (SEQ ID NO: 38) or G₄(SG₄)_(n)(SEQ ID NO: 39), wherein n is an integer between 1 and 10, typicallybetween 2 and 4. A non-immunogenic peptide linker used herein maycomprise glycine-alanine polymers, alanine-serine polymers, and otherflexible peptide linkers. A suitable peptide linker for fusing the firstFab and/or the second Fab to the second Fv region is a glycine-serinepolymers, such as (GGS)₃ (SEQ ID NO: 10).

Peptide linkers can be also derived from immunoglobulin light or heavychain constant domain, such as CLκ or CLλ domains or the CH1 domain, butnot all residues of such a constant domain, for example only the first5-12 amino acid residues. In an embodiment, the peptide linkers is not aimmunoglobulin light or heavy chain constant domain. In an embodiment,the peptide linker is not a CLκ, CLλ, CH1, CH2 or CH3 domain.

Exemplary peptide linkers which may be used in an antigen-bindingmolecule are derived from immunoglobulin light or heavy chain constantdomain are QPKAAP (SEQ ID NO: 12) or ASTKGP (SEQ ID NO: 11). In general,peptide linkers can be derived from immunoglobulin heavy chains of anyisotype, including for example Cy1, Cy2, Cy3, Cy4, Ca1, Ca2, C8, Cs, andOμ.

A peptide linker may also comprise an immunoglobulin hinge (e.g. a humanIgG1 hinge or part thereof) or any peptide derived from such hinge.Preferably, where only a part or portion of an immunoglobulin hinge isused, the truncated hinge may still include one or more of itsinterchain cysteines. The presence of the interchain cysteines allowsfor the formation of a dimeric peptide linker (or hinge region) bydisulfide bridges, in situations where two of such hinge peptide linkersare used. A preferred situation for the use of such disulfide stabilizeddimeric peptide linkers is the fusion of the variable domains of thesecond Fv region to the Fc domain subunits. The presence of adimeric-peptide linker or hinge region additionally promotes andstabilizes the dimerization of the two Fc region subunits present in anantigen-binding molecule according to the present disclosure. Anexemplary human IgG hinge derived peptide linker suited for dimerizationis DKTHTCPPCP (SEQ ID NO: 13).

It is understood that a peptide linker as used herein is not limited toonly one of the aforementioned and exemplified peptide linkers but mycomprise any combination of two or more such linker which are fused toeach other. For instance, a peptide linker as used herein may be builtfrom a glycine-serine polymer and an immunoglobulin hinge derivedsequence in order to retain or obtain the desired activity.

Alternatively, a variety of non-proteinaceous polymers, including butnot limited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may be used as linkers.

The Fc Region

The Fc region of an antigen-binding molecule according to the presentdisclosure consists of a pair of polypeptides comprising heavy chaindomains of a regular immunoglobulin. The Fc region of a regular IgGexists as a dimer, each subunit of which comprises the CH2 and CH3 IgGheavy chain constant domains. The two Fc region subunits are capable ofstable association with each other. Accordingly, in an embodiment, thetwo Fc region subunits of an antigen-binding molecule according to thepresent disclosure are capable of stable association with each other. Inan embodiment, the Fc region of an antigen-binding molecule according tothe present disclosure is an IgG Fc region. In an embodiment, the Fcregion is an IgG1 Fc region. In an embodiment, the Fc region is human.In an embodiment, the Fc region is a human IgG1 Fc region.

The Heterodimeric Fc Region

The two Fc region subunits of an antigen-binding molecule according tothe present disclosure are typically comprised in two non-identicalpolypeptide chains. To improve the yield and purity of the molecule inrecombinant production, it is advantageous to introduce in the Fc regionone or more modifications promoting the association of the twonon-identical polypeptides forming the Fc region subunits. Accordingly,in certain embodiments, the present disclosure provides heterodimericantigen-binding molecules that rely on the use of two different variantFc region subunits that will self-assemble to form a heterodimericmolecule.

In an embodiment, the Fc region of an antigen-binding molecule accordingto the present disclosure comprises one or more modifications promotingthe association of the first and the second Fc region subunit. In anembodiment, the first and second Fc region subunit of an antigen-bindingmolecule according to the present disclosure comprises one or moremodification promoting the association of the first and the second Fcregion subunit. In an embodiment, the first Fc region subunit and secondFc region subunit comprises one or more modification that reducehomodimerization or reduce homodimer formation between two identicalpolypeptide chains comprising the same Fc region subunit.

A modification may be present in the first Fc region subunit and/or thesecond Fc region subunit. In a preferred embodiment, a modification ispresent in the first and second Fc region subunit. In one embodiment,said modification occurs in the CH3 domain of an Fc region subunit. Amodification can be made by altering the nucleic acid encoding thepolypeptides, e.g. by site-specific mutagenesis, or by peptidesynthesis. Several approaches for CH3 modifications in order to promoteheterodimerization have been described, for example in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO2013/157954, WO 2013/096291, which are herein incorporated by reference.

Typically, in the heterodimerization approaches known in the art, theCH3 domain of one polypeptide chain (e.g. immunoglobulin heavy chain)and the CH3 domain of the other polypeptide chain are both engineered ina complementary manner so that the polypeptide comprising one engineeredCH3 domain can no longer homodimerize with another polypeptide chain ofthe same structure. Thereby the polypeptide comprising one engineeredCH3 domain is forced to heterodimerize with the other polypeptidecomprising the CH3 domain, which is engineered in a complementarymanner.

One heterodimerization approach known in the art is the so-called“knobs-into-holes” technology, which is described in detail providingseveral examples in e.g. WO 96/027011, Ridgway, J. B., et al, ProteinEng. 9 (1996) 617-621; Merchant, A. M., et al, Nat. Biotechnol. 16(1998) 677-681; U.S. Pat. Nos. 5,731,168; 7,695,936; WO 98/050431,Carter, J Immunol Meth 248, 7-15 (2001) which are incorporated byreference. The “knobs-into-holes” technology broadly involves: (1)mutating the CH3 domains in each Fc region subunit to promoteheterodimerization; and (2) combining the mutated Fc region subunitsunder conditions that promote heterodimerization. “Knobs” or“protuberances” are typically created by replacing a small amino acid ina parental antibody with a larger amino acid (e.g., T366Y or T366W);“Holes” or “cavities” are created by replacing a larger residue in aparental antibody with a smaller amino acid (e.g., Y407T, T366S, L368Aand/or Y407V) with numbering according EU index.

In an embodiment, the modification present in the Fc region of anantigen-binding molecule according to the present disclosure is a“knobs-into-holes” modification, comprising “knob mutations” in one ofthe two Fc region subunits and “hole mutations” in the othercomplementary Fc region subunit. The knob modifications and holemodifications can be made by altering the nucleic acid encoding thepolypeptides, e.g. by site-specific mutagenesis, or by peptidesynthesis. In an embodiment, the CH3 domain of each Fc region subunit ismodified according to the knobs-into-holes technology.

In an embodiment, in the CH3 domain of the first Fc region subunit, thethreonine residue at position 366 is replaced with a tryptophan residue(T366W) and in the CH3 domain of the second Fc region subunit thetyrosine residue at position 407 is replaced with a valine residue(Y407V) with numbering according EU index. In an embodiment, in the CH3domain of the second Fc region subunit, the threonine residue atposition 366 is replaced with a serine residue (T366S) and the leucineresidue at position 368 is replaced with an alanine residue (L368A) withnumbering according EU index.

In an embodiment, in the CH3 domain of the first Fc region subunit, theserine residue at position 354 is replaced with a cysteine residue(S354C), and in the CH3 domain of the second Fc region subunit thetyrosine residue at position 349 is replaced by a cysteine residue(Y349C) with numbering according EU index based. Introduction of thesetwo cysteine residues results in formation of a disulfide bridge betweenthe two Fc region subunits, further stabilizing the dimer (Carter, JImmunol Methods 248, 7-15 (2001)).

In a more specific embodiment, the present disclosure provides anantigen-binding molecule, wherein in the CH3 domain of first Fc regionsubunit, the threonine residue at position 366 is replaced with atryptophan residue (T366W) and the serine residue at position 354 isreplaced with a cysteine residue (S354C) and in the CH3 domain of thesecond Fc region subunit the tyrosine residue at position 407 isreplaced with a valine residue (Y407V), the threonine residue atposition 366 is replaced with a serine residue (T366S), the leucineresidue at position 368 is replaced with an alanine residue (L368A) andthe tyrosine residue at position 349 is replaced by a cysteine residue(Y349C) with numbering according EU index.

In an alternative embodiment, a modification promoting the associationof the first and the second Fc region subunit comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc regionsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Fc Binding

The Fc region of an immunoglobulin generally confers to the favorablepharmacokinetic properties of antibodies, such as prolonged half-life inserum and to the ability to mediate effector function via binding to Fcreceptors expressed on cells. On the other hand, binding to Fc receptorsmight also results in an undesirable activation of certain cell surfacereceptors leading to unwanted cytokine release and severe side effectsupon systemic administration.

Accordingly, in certain embodiments, the Fc region of an antigen-bindingmolecule according to the present disclosure is engineered to have analtered binding affinity to an Fc receptor and/or to C1q or to havealtered effector function, as compared to a non-engineered Fc region.

Altered effector function can include, but is not limited to, one ormore of the following: altered complement dependent cytotoxicity (CDC),altered antibody-dependent cell-mediated cytotoxicity (ADCC), alteredantibody-dependent cellular phagocytosis (ADCP), altered cytokinesecretion, altered immune complex-mediated antigen uptake byantigen-presenting cells, altered binding to NK cells, altered bindingto macrophages, altered binding to monocytes, altered binding topolymorphonuclear cells, altered direct signaling inducing apoptosis,altered crosslinking of target-bound antibodies, altered dendritic cellmaturation, or altered T cell priming. In particular embodiments, thealtered effector function is one or more selected from the groupconsisting of CDC, ADCC and ADCP. In an embodiment, the altered effectorfunction is ADCC. In an embodiment, the altered effector function isCDC. In an embodiment, the altered effector function is ADCP. In anembodiment, the altered effector function is CDC, ADCC and ADCP.

Altered effector functions are typically achieved by mutating at leastone, preferably both, of the parental Fc domain subunits. Substitutionsthat result in increased binding as well as decreased binding can beuseful. For altering the binding properties of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are preferred.

Decreased Fc Receptor Binding and/or Effector Function

For certain therapeutic situations, it may be desirable to reduce orinhibit the normal binding of the Fc region to one or more or all of theFc receptors and/or binding to a complement component, such as C1q. Forinstance, it may be desirable to reduce or prevent the binding of an Fcregion to one or more or all of the Fcγ receptors (e.g. FcγRI, FcγRIIa,FcγRIIb, FcγRIIIa).

In particular, when an antigen-binding molecule co-engages a receptor ofan immune effector cell, it is advisable to prevent FcγRIIIa binding toabolish or significantly reduce ADCC activity and/or to prevent C1qbinding to eliminate or significantly reduce CDC activity.

The reduced or abolished effector function can include, but is notlimited to, one or more of the following: reduced complement dependentcytotoxicity (CDC), reduced or abolished antibody-dependentcell-mediated cytotoxicity (ADCC), reduced or abolishedantibody-dependent cellular phagocytosis (ADCP), reduced or abolishedcytokine secretion, reduced or abolished immune complex-mediated antigenuptake by antigen-presenting cells, reduced or abolished binding to NKcells, reduced or abolished binding to macrophages, reduced or abolishedbinding to monocytes, reduced or abolished binding to polymorphonuclearcells, reduced or abolished direct signaling inducing apoptosis, reducedor abolished crosslinking of target-bound antibodies, reduced orabolished dendritic cell maturation, or reduced or abolished T cellpriming. In certain embodiments, the reduced or abolished effectorfunction is one or more selected from the group consisting of CDC, ADCCand ADCP. In an embodiment, the reduced or abolished effector functionis ADCC. In an embodiment, the reduced or abolished effector function isCDC. In an embodiment, the reduced or abolished effector function isADCP. In an embodiment, the reduced or abolished effector function isCDC, ADCC and ADCP.

In an embodiment, the Fc region of an antigen-binding molecule accordingto the present disclosure is engineered to have a reduced bindingaffinity to an Fc receptor and/or to C1q and/or to have reduced effectorfunction when compared to a non-engineered Fc region.

In an embodiment, the Fc region of an antigen-binding molecule accordingto the present disclosure is engineered to have reduced effectorfunction when compared to a non-engineered Fc region. In an embodiment,the Fc region of an antigen-binding molecule according to the presentdisclosure comprises one or more amino acid mutation that reduces thebinding affinity of the Fc region to an Fc receptor and/or to C1q and/orreduces the effector function. In general, the same one or more aminoacid mutation(s) is present in each of the two Fc region subunitsforming the Fc region. In an embodiment, the one or more amino acidmutations reduces the binding affinity of the Fc region to an Fcreceptor. Where there is only one amino acid mutation that reduces thebinding affinity of the Fc region to the Fc receptor and/or to C1q, theone amino acid mutation reduces the binding affinity of the Fc region toan Fc receptor and/or to C1q by at least 2-fold, at least 5-fold, or atleast 10-fold and/or reduces the effector function by at least 2-fold,at least 5-fold, or at least 10-fold when compared to the non-engineeredFc region. Where there is more than one amino acid mutation that reducesthe binding affinity of the Fc region to the Fc receptor and/or to C1q,the combination of these amino acid mutations may reduce the bindingaffinity of the Fc region to an Fc receptor and/or to C1q by at least10-fold, at least 20-fold, or at least 50-fold and/or may reduce theeffector function by at least 10-fold, at least 20-fold, or at least50-fold when compared to the non-engineered Fc region.

In an embodiment, the engineered Fc region does substantially not bindto an Fc receptor and/or C1q and/or induce effector function. In anembodiment, the Fc receptor is a human Fc receptor. In one embodiment,the Fc receptor is an activating Fc receptor. In an embodiment, the Fcreceptor is an Fcγ receptor. In an embodiment, the Fc receptor is anactivating human Fcγ receptor, more specifically human FcγRIIIa, FcγRIor FcγRIIa, most specifically human FcγRIIIa.

In an embodiment, the binding affinity of the Fc region to a complementcomponent, in particular the binding affinity to C1q, is reduced orabolished. In an embodiment, the reduced or abolished effector functionis one or more selected from the group of reduced or abolished CDC,reduced or abolished ADCC and reduced or abolished ADCP. In a particularembodiment, the reduced or abolished effector function is reduced ADCC,CDC, and ADCP.

In an embodiment, the Fc region of an antigen-binding molecule accordingto the present disclosure comprises one or more amino acid mutation(s)that reduce(s) the binding affinity of the Fc region to an Fc receptorand/or to C1q and/or reduces the effector function.

In an embodiment, the amino acid mutation is an amino acid substitution.In an embodiment, the Fc region of an antigen-binding molecule accordingto the present disclosure comprises one or more amino acid mutationsthat reduces the binding affinity of the Fc region to an Fc receptorand/or to C1q and/or reduces the effector function, wherein each Fcregion subunit comprises an amino acid substitution at a positionselected from the group of 234, 235, 237, 330 and 331 with numberingaccording EU index.

In an embodiment, each Fc region subunit of an antigen-binding moleculeaccording to the present disclosure comprises an amino acid substitutionat a position selected from the group of L234, L235 and G237 (numberingaccording EU index). In an embodiment, each Fc subunit comprises theamino acid substitutions L234A and L235E with numbering according EUindex. In an embodiment, each Fc region subunit comprises the amino acidsubstitutions L234A, L235E and G237A with numbering according EU index.In an embodiment, each Fc region subunit comprises an amino acidsubstitution at a position selected from the group of 330 and 331 withnumbering according EU index. In an embodiment, each Fc region subunitcomprises an amino acid substitution at the positions 330 and 331 withnumbering according EU index. In an embodiment, the amino acidsubstitution is A330S or P331S.

In an embodiment, the Fc region of an antigen-binding molecule accordingto the present disclosure comprises one or more amino acid mutations ineach Fc region subunit that reduces the binding affinity of the Fcregion to an Fc receptor and/or to C1q and/or reduces the effectorfunction, wherein said one or more amino acid mutations are L234A,L235E, G237A, A330S and P331S.

In an embodiment, the Fc region of an antigen-binding molecule accordingto the present disclosure consists of one or more amino acid mutation ineach Fc region subunit that reduces the binding affinity of the Fcregion to an Fc receptor and/or to C1q and/or reduces the effectorfunction, wherein the one or more amino acid mutations are L234A, L235E,G237A, A330S and P331S. In an embodiment, the Fc region is an IgG1 Fcregion, particularly a human IgG1 Fc region.

Mutant Fc regions or Fc region subunits can be prepared by amino aciddeletion, substitution, insertion or modification using genetic orchemical methods well known in the art. Genetic methods may includesite-specific mutagenesis of the encoding DNA sequence, PCR, genesynthesis, and the like. The correct nucleotide changes can be verifiedfor example by sequencing.

Increased Fc Receptor Binding and/or Effector Function

In certain situations, it may me desirable to enhance or increase Fcreceptor binding and/or C1q binding and/or effector function of anantigen-binding molecule according to the present disclosure.

In an embodiment, the increased or enhanced effector function is one ormore selected from the group of CDC, ADCC, ADCP. In an embodiment, theincreased or enhanced effector function is ADCC. In an embodiment, theincreased or enhanced effector function is CDC. In an embodiment, theincreased or enhanced effector function is ADCP. In an embodiment, theincreased or enhanced effector function is ADCC, ADCP and CDC.Accordingly, in certain embodiments, the Fc region of an antigen-bindingmolecule according to the present disclosure is engineered to have anincreased binding affinity to an Fc receptor and/or to C1q and/or tohave increased effector function when compared to the non-engineered Fcregion.

Accordingly, in an embodiment, the Fc region of an antigen-bindingmolecule according to the present disclosure is engineered to have anincreased binding affinity to an Fc receptor when compared thenon-engineered Fc region. In an embodiment, the Fc region of anantigen-binding molecule according to the present disclosure isengineered to have an increased binding affinity to C1q when comparedthe non-engineered Fc region. In certain embodiments, the Fc region ofan antigen-binding molecule according to the present disclosure isengineered to have increased effector function when compared to thenon-engineered Fc region.

In an embodiment, the Fc region of an antigen-binding molecule accordingto the present disclosure comprises one or more amino acid mutations ineach Fc region subunit that increase the binding affinity of the Fcregion to an Fc receptor and/or to C1q and/or increases the effectorfunction. Increased binding affinity may be an increase in the bindingaffinity of the Fc region to the Fc receptor and/or C1q by at least2-fold, at least 5-fold, or at least 10-fold when compared to thenon-engineered Fc region. Typically, the same amino acid mutations arepresent in each of the two Fc region subunits. In an embodiment, the oneor more amino acid mutation increases the binding affinity of the Fcregion to an Fc receptor when compared to the non-engineered Fc region.In an embodiment, the one or more amino acid mutation increases thebinding affinity of the Fc region to C1q when compared to thenon-engineered Fc region. Examples of amino acid mutations, which resultin an increase in binding affinity of an Fc region to an Fc receptorand/or C1q are described in WO 2000/042072 or WO 2004/099249, which areincorporated by reference.

Typically, an amino acid mutation that increases the binding affinity ofthe Fc region to an Fc receptor and/or to C1q and/or increases effectorfunction is an amino acid substitution.

In embodiments, where there is only one amino acid mutation thatincreases the binding affinity of the Fc region to the Fc receptorand/or to C1q, the one amino acid mutation may increase the bindingaffinity of the Fc region to an Fc receptor and/or to C1q by at least2-fold, at least 5-fold, or at least 10-fold and/or may increase theeffector function by at least 2-fold, at least 5-fold, or at least10-fold when compared to the non-engineered Fc region. In embodiments,where there is more than one amino acid mutation that increases thebinding affinity of the Fc region to the Fc receptor and/or to C1q, thecombination of these amino acid mutations may increase the bindingaffinity of the Fc region to an Fc receptor and/or C1q by at least10-fold, at least 20-fold, or at least 50-fold and/or may increase theeffector function by at least 10-fold, at least 20-fold, or at least50-fold when compared to the non-engineered Fc region.

In an embodiment, the Fc receptor is a human Fc receptor. In anembodiment, the Fc receptor is an activating Fc receptor. In anembodiment, the Fc receptor is a Fcγ receptor. In an embodiment, the Fcreceptor is an activating human Fcγ receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa. In an embodiment, the Fc receptor isselected from the group of FcγRIIIa, FcγRI and FcγRIIa. In a particularembodiment, the Fc receptor is FcγRIIIa.

In an embodiment, the increased effector function is one or moreselected from the group of increased ADCC, increased CDC and increasedADCP. In an embodiment, the increased effector function is increasedADCC.

In Vitro Methods to Assess Binding to Fc Receptors or to Assess ImmuneEffector Function

Binding of the Fc region to Fc receptors can be easily determined e.g.by ELISA, or by Surface Plasmon Resonance (SPR) using standardinstrumentation such as a BIAcore instrument (GE Healthcare), and Fcreceptors may be obtained by recombinant expression. Alternatively, thebinding affinity of Fc regions may be evaluated using cell lines knownto express particular Fc receptors, such as NK cells expressing FcγIIIareceptor. Effector function of an Fc region can be measured by methodsknown in the art. Suitable in vitro assays to assess ADCC activity of amolecule of interest are for instance described in WO2012130831. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g. in an animal model such as that disclosed in Clynes etal., Proc Natl Acad Sci USA 95, 652-656 (1998). To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al.,Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743(2004)). C1q binding assays (such as ELISA) may be carried out todetermine whether an antigen-binding molecule is able to bind C1q andhence has CDC activity (WO 2006/029879 and WO 2005/100402).

Target Antigens

The novel antigen-binding molecules according to the present disclosureare suited for targeting a variety of antigens and are particularlysuited for targeting different antigens simultaneously.

“Antigen” or “target antigen” as used herein refers to any molecule ofinterest that specifically binds to one of the Fv regions present in anantigen-binding binding molecule according to the present disclosure.Typically, an antigen is a peptide, a protein or any other proteinaceousmolecule. Alternatively, an antigen may be any other organic orinorganic molecule, such as carbohydrate, fatty acid, lipid, dye orflourophor.

The ability of an antigen-binding molecule according to the presentdisclosure to specifically bind to an target antigen can be measuredeither through an enzyme-linked immunosorbent assay (ELISA) or othertechniques familiar to one of skill in the art, e.g. surface plasmonresonance technique (analyzed on a BIACORE T100 system) (Liljeblad, etal., Glyco J 17, 323-329 (2000)), and traditional binding assays(Heeley, Endocr Res 28, 217-229 (2002)).

Competition assays may be used to identify an antibody, antibodyfragment, antigen-binding domain or variable domain that cross-competeswith a reference antibody for binding to a specific antigen or epitope.“Cross competes” means the ability of an antibody, antibody fragment orantigen-binding molecules to interfere with the binding of otherantibodies, antibody fragments or antigen-binding molecules to aspecific antigen in a standard competitive binding assay. The ability orextent to which an antibody, antibody fragment or antigen-bindingmolecule is able to interfere with the binding of another antibody,antibody fragment or antigen-binding molecule to a specific antigen,and, therefore whether it can be said to cross-compete according to thepresent disclosure, can be determined using standard competition bindingassays. One suitable assay involves the use of the Biacore technology(e.g. by using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden)),which can measure the extent of interactions using surface plasmonresonance technology. Another assay for measuring cross-competing usesan ELISA-based approach. A high throughput process for “epitope binning”antibodies based upon their cross-competition is described inInternational Patent Application No. WO 2003/48731. In certainembodiments, such a competing antibody or antigen-binding-molecule bindsto the same epitope (e.g. a linear or a conformational epitope) that isbound by the reference antibody or antigen-binding molecule.

Accordingly, an antigen-binding molecule according to the presentdisclosure preferably targets two or more, even more preferably, twodifferent antigens (e.g. a first and a second antigen). The two antigenscan be expressed on the surface of one cell or can be expressed on thesurface of different cells. The ability to target two different antigenswith different valency (e.g. one antigen monovalently and one antigenbivalently) is a particular useful aspect of an antigen-binding moleculeaccording to the present disclosure. As outlined before, for some immunereceptors (such as the CD3 signaling receptor on T cells) receptoractivation only upon binding to the co-target (e.g. a tumor-associatedantigen) is desired, because non-specific cross-linking in a clinicalsetting can result in a life-threatening cytokine storm. By binding suchimmune receptors monovalently, receptor activation will only occur inresponse of cross-linking to the co-target.

In an embodiment, the first and/or the second antigen is an antigenassociated with a pathological condition, such as an antigen presentedon a tumor cell, on a virus-infected cell, or an antigen expressed at asite of inflammation. In an embodiment, the first or second antigen ispreferably an antigen expressed on immune cells, such as T-cells. Othersuitable antigens include cell surface antigens (such as cell surfacereceptors), antigens free in blood serum, and/or antigens in theextracellular matrix. In an embodiment, the antigen is a human antigen.

In an embodiment, the first antigen is a tumor-associated antigen,specifically an antigen presented on a tumor cell or a cell of the tumorstroma. In an embodiment, the first antigen is a HLA-restricted peptide.In an embodiment, the first antigen is a peptide/HLA-A0201 complex. Theterm “HLA-A0201” refer to a specific HLA serotype. HLA-A0201 is aheterodimeric protein, comprising an alpha chain and a beta chain. In anembodiment, the peptide/HLA-A0201 complex is expressed on a cancer cell.In an embodiment, the peptide/HLA-A0201 complex is specific for a cancercell. In an embodiment, the first antigen is a cancer specificHLA-restricted peptide expressed on the surface of a cancer cell

Non-limiting examples of (tumor-associated) antigens include antigenssuch as AR, AGR2, A1G1, AKAP1, AKAP2, ANGPT1, ANGPT2, ANPEP, ANGPTL3,APOC1, ANGPTL4, AITGAV, AZGP1, BMP6, BRCA1, BAD, BAG1, BCL2, BL6R, BA2,BPAG1, CDK2, CD52, CD20, CD19, CD3, CD4, CD8, CD164, CDKN1A, CDKN1B,CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, CDK3, CDK4, CDK5, CDK6, CDK7,CDK9, CLDN3, CLN3, CYB5, CYC1, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, CHGB, CDH20, CDH7, CDH8, CDH9, CD44, CDH1, CDH10, CDH19, CDH20,CDH7, CDH9, CDH13, CDH18, CDH19, CANT1, CAV1, CDH12, CD164, COL6A1,CCL2, CDH5, COL18A1, CHGA, CHGB, CLU, COLIA1, COL6A1, CCNA1, CCNA2,CCND1, CCNE1, CCNE2, COL6A1, CTNNB1, CTSB, CLDN7, CLU, CD44APC, COL4A3,DSfHA, DAB2JP, DES, DNCL1, DD2, DL2, EL24, EGF, E2F1, EGFR, ENO1, ERBB2,ESR1, ESR2, EL2, EStHA, ELAC2, ENO2, ENO3, ERBB2, ESR1, ESR2, EDG1,EFNA1, EFNA3, EFNB2, EPHB4, ESR1, ESR2, EGF, ERK8, EL12A, EL1A, EL24,ENHA, ELK, ECGF1, EREG, EDG1, ENG, E-cadherin, FGF1, FGF10, FGF11,FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21,FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FASN, FLJ12584,FLJ25530, F1GF, FLT1, FGFR3, F3, FOSL1, FLRT1, IL12A, IL1A, IL1B, IL2,INHA, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, IGF1R, IL2, IGFBP6,IL1A, IL1B, IGFBP3, IGFBP6, INSL4, IL6ST, ITGA6, IGF1, IGF2, INSL3,INSL4, IFNA1, IFNB1, IFNG, IL1B, IL6, IGFBP2, IL2RA, IL6, IGF1, IGF2,IGFBP3, IGFBP6, ITGA1, IGF1, ITGA6, ITGB4, INSL3, INSL4, IL29, IL8,ITGB3, GRP, GNRH1, GAGEB1, GAGEC1, GGT1, GSTP1, GATA3, GABRP, GNAS1,GSN, H1P1, HUMCYT2A, HGF, JAG1, JUN, LAMA5, S100A2, SCGB1D2, SCGB2A1,SCGB2A2, SPRR1B, SHBG, SERP1NA3, SHBG, SLC2A2, SLC33A1, SLC43A1, STEAP,STEAP2, SERP1NF1, SERPINB5, SERPINE1, STAB1, TGFA, TGFB1, TGFB2, TGFB3,TNF, TNFSF10, TGFB1I1, TP53, TPM1, TPM2, TRPC6, TGFA, THBS, TEE,TNFRSF6, TNFSF6, TOP2A, TP53, THBS1, THBS2, THBS4, TNFAIP2, TP53, TEK,TGFA, TGFB1, TGFB2, TGFBR1, TGFA, TEV1P3, TGFB3, TNFA1P2, 1TGB3, THBS1,THBS2, VEGF, VEGFC, ODZ1, PAWR, PLG, PAP, PCNA, PRKCQ, PRKD1, PRL,PECAM1, PF4, PROK2, PRL, PAP, PLAU, PRL, PSAP, PART1, PATE, PCA3, P1AS2,PGF, PGR, PLAU, PGR, PLXDCI, PTEN, PTGS2, PDGF, MYC, MMP2, MMP9, MSMB,MACMARCK5, MT3, MUC1, MAP2K7, MKi67, MTSS1, M1B1, MDK, NOX5, NR6A1,NR1H3, NR1I3, NR2F6, NR4A3, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2, NR2E1,NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1, NR5A2, NR6A1,NROB1, NROB2, NR1D2, NR1D1, NTN4, NRP1, NRP2, NGFB, NGFR, NME1, KLK6,KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, K6HF,KA2, KRT2A, KLK6, KLK3, KRT1, KDR, KLK5, KRT19, KLF5, KRT19, KRTHB6,RARB, RAC2, and ROBO2.

In an embodiment, the first or second antigen is selected from the groupof HER2 and CD3. In an embodiment, the first antigen is HER2,particularly human HER2. In an embodiment, the first antigen is CD3,particularly human CD3. In an embodiment, the second antigen is HER2,particularly human HER2. In an embodiment, the second antigen is CD3,particularly human CD3. In an embodiment, the first and/or third Fvregion can compete with monoclonal antibody Trastuzumab for binding toan epitope of HER2.

In an embodiment, the first and/or third Fv region specifically binds toHER2 and comprises a VH sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8 and a VL sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 9, or variants thereof that retainfunctionality.

In an embodiment, the first or second antigen is expressed on an immunecell. In an embodiment, the second antigen is expressed on a T-cell. Inan embodiment, the first antigen is expressed on a T-cell.

In an embodiment, the first or second antigen is selected from the groupconsisting of CD137 and CD3. In an embodiment, the first antigen isselected from the group consisting of CD137 and CD3. In an embodiment,the second antigen is selected from the group consisting of CD137 andCD3. In an embodiment, the first antigen is CD3, in particular humanCD3. In an embodiment, the second antigen is CD3, in particular humanCD3. In an embodiment, CD3 is bound monovalently by an antigen-bindingmolecule of the present disclosure.

CD3 is a proven T cell stimulating antigen with therapeutic relevance.Binding to CD3 mimics the T-cell receptor (TCR) leading to T-cellactivation. Using CD3 binding molecules in a multispecificantigen-binding molecule in such a way that the target cells and theT-cells are bridged via the multispecific antigen-binding moleculeresulting in the formation of an immunological synapse, the effector Tcells are able to kill the target cell directly. It is known thatefficacy and safety of such molecules with co-engagement of CD3 ismainly driven by the binding valency, the affinity of both specificitiesand the format used. The binding format should engage CD3 monovalentlywith moderate to low binding affinity to reduce the potential risk forside effects as discussed before. In order to increase the efficacywithout the need for increasing affinity, the epitope of the targetantigen (e.g. the first antigen) and of CD3 (e.g. the second antigen)should be in close proximity to enable the immunological synapse(Bluemel C., Cancer Immunol. Immunother. 2010 August; 59(8):1197-209).In addition, the format should further supports low frequencies ofdosage in such a way that a usual IgG pharmacokinetic is achieved e.g.via an Fc region.

In an embodiment of the present disclosure, the second antigen is CD3,particularly human or cynomolgus CD3, most particularly human CD3. In anembodiment, the second antigen is the epsilon subunit of CD3. In anembodiment, the second antigen is the epsilon subunit of CD3 comprisingSEQ ID NO: 1.

In an embodiment, the second Fv region of an antigen-binding moleculeaccording to the present disclosure specifically binds to CD3,particularly human or cynomolgus CD3, most preferably human CD3. In anembodiment, CD3 is bound monovalently by an antigen-binding moleculeaccording to the present disclosure. In an embodiment, the second Fvregion can compete with a monoclonal antibody specific for CD3 forbinding to an epitope of CD3. In an embodiment, the second Fv region cancompete with any one of the antibodies specific for CD3 as described inTables 3-5 for binding to an epitope of CD3.

In an embodiment, the second Fv region present in an antigen-bindingmolecule according to the present disclosure can compete with themonoclonal antibody comprising the VH of SEQ ID NO: 4 and the VL of SEQID NO: 5 for binding to an epitope of CD3.

In an embodiment, the second Fv region can compete with the monoclonalantibody comprising the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 3for binding to an epitope of CD3.

In an embodiment, the second Fv region can compete with the monoclonalantibody comprising the VH of SEQ ID NO: 6 and the VL of SEQ ID NO: 7for binding an epitope of CD3.

In a further embodiment, the second Fv region specific for CD3 comprisesa VH that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID SEQ ID NO: 2 and a VL sequence that is at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 orvariants thereof that retain functionality.

In a further embodiment, the second Fv region that is specific for CD3comprises a VH sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID SEQ ID NO: 4 and a VL sequence thatis at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 5 or variants thereof that retain functionality.

In a further embodiment, the second Fv region that is specific for CD3comprises a VH sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID SEQ ID NO: 6 and a VL sequence thatis at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 7 or variants thereof that retain functionality.

In an embodiment of to the present disclosure, an antigen-bindingmolecule according to the present disclosure is capable of simultaneousbinding to a target cell antigen, particularly a tumor-associatedantigen expressed on a cancer cell and CD3 expressed on an immuneeffector cell. In one such embodiment, the target antigen is boundbivalently and CD3 is bound monovalently.

In an embodiment, an antigen-binding molecule according to the presentdisclosure is capable of crosslinking a T-cell and a target cell bysimultaneous binding to a target cell antigen and CD3. In an embodiment,such simultaneous binding results in lysis of the target cell,particularly lysis of a tumor cell. In one embodiment, such simultaneousbinding results in activation of the T-cell. In an embodiment, thesimultaneous binding results in a cellular response of a T-lymphocyte,particularly a cytotoxic T-lymphocyte, selected from the group of:proliferation, differentiation, cytokine secretion, cytotoxic effectormolecule release, cytotoxic activity, and expression of activationmarkers. In an embodiment, binding of an antigen-binding moleculeaccording to the present disclosure to CD3 without simultaneous bindingto the target cell antigen does not result in T-cell activation. In anembodiment, the antigen-binding molecule is capable of re-directingcytotoxic activity of a T-cell to a target cell. In a particularembodiment, the re-direction is independent of MHC-mediated peptideantigen presentation by the target cell and and/or specificity of theT-cell. Particularly, a T-cell according to any of the embodimentsaccording to the present disclosure is a cytotoxic T-cell. In someembodiments, the T-cell is a CD4+ or a CD8+ T cell, particularly a CD8+T cell.

A format realized in the antigen-binding molecules of the presentdisclosure enables bivalent binding to a first antigen and monovalentbinding to a second antigen and as such combines high affinity bindingand avidity effects for the first antigen resulting in a significantdifference in binding affinity between CD3 and the target antigen.

An antigen-binding molecule according to the present disclosure areparticularly beneficial for targeting different antigens. However, insome cases it may be beneficial to target only one antigen and as suchhave specificity for the same antigen.

Nucleic Acids

The present disclosure provides a nucleic acid composition comprising anucleic acid sequence or a plurality of nucleic acid sequences encodingan antigen-binding molecule according to the present disclosure. Anantigen-binding molecule according to the present disclosure may consistof one, two, three, four, or even more polypeptides. Each of saidpolypeptides may be encoded by the same or by different nucleic acidsequences. Likewise, the nucleic acid sequences encoding said individualpolypeptides of an antigen-binding molecule according to the presentinvention may be present on the same or on different vectors.

In an embodiment, the present disclosure provides a nucleic acidcomposition comprising a nucleic acid sequence or a plurality of nucleicacid sequences encoding an antigen-binding molecule according to thepresent disclosure. In an embodiment, the present disclosure provides anucleic acid composition comprising a nucleic acid sequence or aplurality of nucleic acid sequences encoding any of the antigen-bindingmolecules described in Tables 9-13. In an embodiment, the nucleic acidcomposition is an isolated nucleic acid composition.

In an embodiment, a first nucleic acid sequence encodes a polypeptidecomprising from the N-terminus to the C-terminus the heavy or lightchain of the first Fab, the VH or VL of the second Fv region and thefirst Fc region subunit. In one such embodiment, a second nucleic acidencodes a polypeptide comprising the complementary heavy or light chainof the first Fab. In one such embodiment, a third nucleic acid encodes apolypeptide comprising from the N-terminus to the C-terminus thecomplementary VH or VL of the second Fv region and the second Fc regionsubunit. In an alternative embodiment, a third nucleic acid encodes apolypeptide comprising from the N-terminus to the C-terminus the heavyor light chain of the second Fab, the complementary VH or VL of thesecond Fv region and the second Fc region subunit. In one suchembodiment, a fourth nucleic acid sequence encodes a polypeptidecomprising the complementary light or heavy chain of the second Fab.

The polypeptides encoded by the nucleic acid sequence or the pluralityof nucleic acid sequences may associate after expression through, e.g.,disulfide bonds or other means to form a functional antigen-bindingmolecule as described herein. For example, the light chain of the firstFab may be encoded by a separate nucleic acid sequence than the portionof an antigen-binding molecule comprising the heavy chain of the firstFab. When co-expressed, the light chain of the first Fab will associatewith the heavy chain of the first Fab to form the first Fab comprising afirst Fv region. In another example, the portion of an antigen-bindingmolecule comprising the first Fc region subunit could be encoded by aseparate nucleic acid sequence than the portion of an antigenbinding-molecule comprising the second Fc region subunit. Whenco-expressed, the two Fc region subunits will associate to form thedimeric Fc region of an antigen-binding molecule according to thepresent disclosure.

In an embodiment, the present disclosure is directed to a nucleic acidsequence or a plurality of nucleic acid sequences encoding anantigen-binding molecule according to the present disclosure, whereinthe nucleic acid sequence or the plurality of nucleic acid sequencesencodes for the individual polypeptides of the antigen-binding molecule.Polypeptides forming the exemplified antigen-binding molecules accordingto the present disclosure are described in Tables 9-13.

Vector

In an embodiment, the present disclosure provides a vector compositioncomprising a vector or a plurality of vectors comprising a nucleic acidsequence composition according to the present disclosure. In anembodiment, the present disclosure provides a vector compositioncomprising a vector or a plurality of vectors comprising a nucleic acidsequence or plurality of nucleic acid sequences encoding anantigen-binding molecule according to the present disclosure. In anembodiment, the present disclosure provides a vector compositioncomprising a vector or a plurality of vectors comprising a nucleic acidsequence or plurality of nucleic acid sequences encoding anantigen-binding molecule as described in Tables 9-13. In certainembodiments, the vector is an expression vector.

Host Cell

In certain embodiments, the present disclosure provides a host cellcomprising a vector composition comprising a vector or a plurality ofvectors comprising a nucleic acid composition comprising a nucleic acidsequence or plurality of nucleic acid sequences encoding anantigen-binding molecule according to the present disclosure. In anembodiment, the present disclosure refers to a host cell comprising avector composition comprising a vector or a plurality of vectorscomprising a nucleic acid composition comprising the nucleic acidsequence or plurality of nucleic acid sequences encoding anantigen-binding molecule as described in Tables 9-13.

Host cells suitable for replicating and for supporting expression of anantigen-binding molecule according to the present disclosure are wellknown in the art. Such host cells may be transfected or transduced asappropriate with the particular expression vector(s) and largequantities of vector containing cells can be grown for seeding largescale fermenters to obtain sufficient quantities of such anantigen-binding molecule for clinical applications. Standardtechnologies are known in the art to express foreign genes in thesesystems. In general, such steps typically include transforming ortransfecting a suitable host cell with a nucleic acid composition or avector composition, which encodes the individual polypeptides of anantigen-binding molecule according to the present disclosure. Further,such steps typically include culturing the host cells under conditionssuitable for the proliferation (multiplication, growth) of the hostcells and a culturing step under conditions suitable for the production(expression, synthesis) of the encoded polypeptides.

Production

Methods to produce antibodies or antigen-binding molecules as disclosedherein are well known in the art (see e.g. Harlow and Lane, “Antibodies,a laboratory manual”, Cold Spring Harbor Laboratory, 1988). Anantigen-binding molecule according to the present disclosure may beobtained, for example, by solid-state peptide synthesis or recombinantproduction. For recombinant production, one or more nucleic acidsequences encoding an antigen-binding molecule according to the presentdisclosure are isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequences for anantigen-binding molecule according to the present disclosure along withappropriate transcriptional/translational control signals. Such methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, N.Y (1989). The vectors can be introduced into theappropriate host cells such as prokaryotic (e.g., bacterial) oreukaryotic (e.g., yeast or mammalian) cells by methods well known in theart (see, e.g., “Current Protocol in Molecular Biology”, Ausubel et al.(eds.), Greene Publishing Assoc. and John Wiley Interscience, New York,1989 and 1992). Numerous cloning vectors are known to those of skill inthe art, and the selection of an appropriate cloning vector is a matterof choice. The coding sequences can be placed under the control of apromoter, ribosome binding site (for bacterial expression) and,optionally, an operator, so that the DNA sequence encoding the desiredprotein or polypeptide is transcribed into RNA in the host celltransformed by a vector or vectors containing this expression construct.The coding sequence may or may not contain a signal peptide or leadersequence. Depending on the expression system and host cell selected, anantigen-binding molecule according to the present disclosure is producedby growing host cells transformed by expression vectors described beforeunder conditions whereby the protein of interest is expressed. Theprotein is then isolated from the host cells and purified. If theexpression system secretes the protein into growth media, the proteincan be purified directly from the media. If the protein is not secreted,it is isolated from cell lysates or recovered from the cell membranefraction. The selection of the appropriate growth conditions andrecovery methods are within the skill of the art.

It should be noted that an antigen-binding molecule according to thepresent disclosure is not a naturally occurring protein. Typically, anantigen-binding molecule according to the present disclosure is arecombinant, synthetic or semi-synthetic protein.

In an embodiment, a method of producing a antigen-binding moleculeaccording to the present disclosure is provided, wherein the methodcomprises culturing a host cell comprising vector composition comprisinga vector or a plurality of vectors comprising a nucleic acid sequence orplurality of nucleic acid sequences encoding an antigen-binding moleculeaccording to the present disclosure, under conditions suitable forexpression of an antigen-binding molecule, and recovering anantigen-binding molecule from the host cell or host cell culture medium.

In embodiments, the methods for the production of antigen-bindingmolecules according to the present disclosure further comprise the stepof isolating the produced antigen-binding molecules from the host cellsor medium. An antigen-binding molecule recovered as described herein maybe purified techniques know in the art, such as high performance liquidchromatography (HPLC), ion exchange chromatography, gel electrophoresis,affinity chromatography, size exclusion chromatography, and the like.The conditions used to purify a particular protein will depend, in part,on factors such as net charge, hydrophobicity, hydrophilicity etc., andwill be apparent to those having skill in the art. For affinitychromatography purification an antibody, ligand, receptor or antigen canbe used to which an antigen-binding molecule binds. For example, foraffinity chromatography purification of antigen-binding moleculesaccording to the present disclosure, a matrix with protein A or proteinG may be used. The purity of an antigen-binding molecule can bedetermined by any of a variety of well-known analytical methodsincluding gel electrophoresis, high-pressure liquid chromatography, andthe like.

Fusion Proteins

An antigen-binding molecule according to the present disclosure may ormay not be fused to one or more other moieties. Such a fusion proteinmay be prepared in any suitable manner, including genetically orchemically approaches. Said linked moieties may contain secretory orleader sequences, sequences that aid detection, expression, separationor purification, or sequences that confer to increased proteinstability, for example, during recombinant production. Non-limitingexamples of potential moieties include beta-galactosidase,glutathione-S-transferase, luciferase, a T7 polymerase fragment, asecretion signal peptide, an antibody or antibody fragment, a toxin, areporter enzyme, a moiety being capable of binding a metal ion like apoly-histidine tag, a tag suitable for detection and/or purification, ahomo- or heteroassociation domain, a moiety which increases solubilityof a protein, or a moiety which comprises an enzymatic cleavage site.Accordingly, an antigen-binding molecule according to the presentdisclosure may optionally contain one or more moieties for binding toother targets or target proteins of interest. It should be clear thatsuch further moieties may or may not provide further functionality to anantigen-binding molecule according to the present disclosure and may ormay not modify the properties of an antigen-binding molecule accordingto the present disclosure. The polypeptides according to the presentdisclosure may be fused by linkers as defined herein.

Functionality

An antigen-binding molecule according to the present disclosure may beused for the prevention and treatment of diseases, which are mediated bybiological pathways in which a target antigen of interest is involved.This may be achieved for instance by inhibiting the interaction betweena target antigen and its cognate receptor or natural binding partner.The biological activity of an antigen-binding molecule according to thepresent disclosure can be measured by various assays known in the art,including those described in Examples 3-4 disclosed herein. Methods forassaying functional activity may utilize binding assays, such as theenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),fluorescence activated cell sorting (FACS) and other methods that arewell known in the art (see Hampton, R. et al. (1990; Serological Methodsa Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al.(1983; J. Exp. Med. 158:1211-1216). Alternatively, assays may test theability of an antigen-binding molecule in eliciting a biologicalresponse because of binding to a biological target antigen, either invivo or in vitro. Biological activities may for example include theinduction of proliferation of T cells, the induction of signaling in Tcells, the induction of expression of activation markers in T cells, theinduction of cytokine secretion by T cells, the inhibition of signalingin target cells such as tumor cells or cells of the tumor stroma, theinhibition of proliferation of target cells, the induction of lysis oftarget cells, and the induction of tumor regression and/or theimprovement of survival.

In an embodiment, the present disclosure provides a method for inducinglysis of a cancer cell, comprising contacting said cancer target cell inthe presence of a cytotoxic T-cell with an antigen-binding moleculeaccording to the present disclosure.

In an embodiment, the present disclosure provides a method forinhibition of signaling in cancer cells comprising contacting saidcancer cells in the presence of a cytotoxic T-cell with anantigen-binding molecule according to the present disclosure.

In an embodiment, the present disclosure provides a method forinhibition of proliferation of cancer cells, comprising contacting saidcancer cells in the presence of a cytotoxic T-cell with anantigen-binding molecule according to the present disclosure.

In an embodiment, the present disclosure provides a method for killing acancer antigen high expressing cells but not cancer antigen lowexpressing cells, comprising contacting said cancer antigen highexpressing cells in the presence of a cytotoxic T-cell with anantigen-binding molecule according to the present disclosure.

In an embodiment, the present disclosure provides a method for inducinga cellular response in cytotoxic T-cells, comprising contacting saidcytotoxic T-cell in the presence of a cancer cell with anantigen-binding molecule according to the present disclosure. In anembodiment, said cellular response is selected from the group consistingof: proliferation, differentiation, cytokine secretion, cytotoxiceffector molecule release, cytotoxic activity, and expression ofactivation markers.

In an embodiment, the present disclosure provides a method for inducinghuman T-cell proliferation in the presence of cancer cells, comprisingcontacting said cancer cell in the presence of a T-cell with aantigen-binding molecule according to the present disclosure

In an embodiment, the present disclosure provides a method forstimulating a primary T-cell response in the presence of cancer cells,comprising contacting said cancer cells in the presence of said T-cellwith an antigen-binding molecule according to the present disclosure.

In an embodiment, the present disclosure provides a method forre-directing cytotoxic activity of a T-cell to a cancer cell, comprisingcontacting said cancer cells in the presence of said T-cell with anantigen-binding molecule according to the present disclosure.

In an embodiment, the present disclosure provides the use of anantigen-binding molecule according to the present disclosure for thetreatment of cancer that is positive for a cancer associated antigen ina subject, comprising:

-   -   (a) selecting a subject who is afflicted with a cancer,    -   (b) collecting one or more biological samples from the subject,    -   (c) identifying the cancer associated antigen expressing cancer        cells in the one or more samples; and    -   (d) administering to the subject an effective amount of an        antigen-binding molecule according to the present disclosure.

Diagnostics

In an embodiment, the present disclosure provides the use of anantigen-binding molecule according to the present disclosure for thediagnosis of a disease. In an embodiment, the present disclosureprovides the use of an antigen-binding molecule according to the presentdisclosure for the detection of an antigen. In an embodiment, thepresent disclosure provides a method for detecting an antigen in asubject or a sample, comprising the step of contacting said subject orsample with an antigen-binding molecule according to the presentdisclosure. In an embodiment, the present disclosure provides a methodfor diagnosing a disease in a subject, comprising the step of contactingsaid subject or sample with an antigen-binding molecule according to thepresent disclosure.

Therapeutic Methods

An antigen-binding molecule according to the present disclosure may beused in therapeutic methods. An antigen-binding molecule according tothe present disclosure may be used for the treatment of cancer. In anembodiment, the present disclosure provides a method for the treatmentof a disease. In an embodiment, the present disclosure provides anantigen-binding molecule according to the present disclosure for thetreatment of a disease. In an embodiment, the present disclosureprovides an antigen-binding molecule according to the present disclosurefor use in the treatment of a disease. In an embodiment, the presentdisclosure provides an antigen-binding molecule according to the presentdisclosure for use in the treatment of a disease in an individual inneed thereof. In an embodiment, the present disclosure provides the useof an antigen-binding molecule according to the present disclosure forthe manufacture of a medicament. In an embodiment, the presentdisclosure provides an antigen-binding molecule according to the presentdisclosure for use as a medicament. In an embodiment, the presentdisclosure provides an antigen-binding molecule according to the presentdisclosure for use as a medicament for the treatment of a disease in anindividual in need thereof. In an embodiment, the disease is associatedwith the undesired presence of an antigen. In an embodiment, the diseaseto be treated is a proliferative disease. In a particular embodiment,the disease is cancer.

Non-limiting examples of cancers include bladder cancer, brain cancer,head and neck cancer, pancreatic cancer, lung cancer, breast cancer,ovarian cancer, uterine cancer, cervical cancer, endometrial cancer,esophageal cancer, colon cancer, colorectal cancer, rectal cancer,gastric cancer, prostate cancer, blood cancer, skin cancer, squamouscell carcinoma, bone cancer, and kidney cancer.

In an embodiment, the present disclosure provides an antigen-bindingmolecule according to the present disclosure for use in a method oftreating a subject or individual having a disease comprisingadministering to the subject a therapeutically effective amount of anantigen-binding molecule according to the present disclosure. In anembodiment, the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent. The subject or individual in need of treatment istypically a mammal, more specifically a human. For use in therapeuticmethods, an antigen-binding molecule according to the present disclosurewould be formulated, dosed, and administered in a way consistent withgood medical practice.

In an embodiment, the present disclosure provides a method for inductionof tumor regression in a patient who has cancer, comprisingadministering to said subject, a therapeutically effective amount of anantigen-binding molecule according to the present disclosure.

In an embodiment, the present disclosure provides a method for improvingsurvival of a subject who has cancer, comprising administering to saidsubject, a therapeutically effective amount of an antigen-bindingmolecule according to the present disclosure.

In an embodiment, the present disclosure provides a method foreliciting, stimulating or inducing an immune response in a subject whohas cancer, comprising administering to said subject, a therapeuticallyeffective amount of an antigen-binding molecule according to the presentdisclosure.

In an embodiment, the present disclosure provides a method for enhancingor inducing anti-cancer immunity in a subject who has cancer, comprisingadministering to said subject, a therapeutically effective amount of anantigen-binding molecule according to the present disclosure.

Pharmaceutical Compositions

In an embodiment, the present disclosure provides a pharmaceuticalcomposition comprising an antigen-binding molecules according to thepresent disclosure and at least one pharmaceutically acceptable carrier.The pharmaceutical compositions may further comprise at least one otherpharmaceutically active compound. The pharmaceutical compositionaccording to the present disclosure can be used in the diagnosis,prevention and/or treatment of diseases associated with a target antigenof interest.

In particular, the present disclosure provides a pharmaceuticalcompositions comprising an antigen-binding molecules according to thepresent disclosure that is suitable for prophylactic, therapeutic and/ordiagnostic use in a mammal, more particular in a human. In general, anantigen-binding molecule according to the present disclosure may beformulated as a pharmaceutical composition comprising at least oneantigen-binding molecule according to the present disclosure and atleast one pharmaceutically acceptable carrier, diluent or excipientand/or adjuvant, and optionally one or more further pharmaceuticallyactive compounds. Such a formulation may be suitable for oral,parenteral, topical administration or for administration by inhalation.

In particular, an antigen-binding molecule according to the presentdisclosure may be used in combination with one or more pharmaceuticallyactive compounds that are or can be used for the prevention and/ortreatment of the diseases in which a target antigen of interest isinvolved, as a result of which a synergistic effect may or may not beobtained. Examples of such compounds, as well as routes, methods andpharmaceutical formulations or compositions for administering them willbe clear to the clinician. In an embodiment, the present disclosureprovides a pharmaceutical composition comprising an antigen-bindingmolecule according to the present disclosure for use in the preventionand/or treatment of a disease associated with the undesired presence ofa target antigen specifically. In an embodiment, the present disclosureprovides a pharmaceutical composition comprising an antigen-bindingmolecule according to the present disclosure for the use as amedicament. In an embodiment, the present disclosure provides apharmaceutical composition comprising an antigen-binding moleculeaccording to the present disclosure for use in the prevention and/ortreatment of autoimmune diseases, inflammatory diseases, cancer,vascular diseases, infectious diseases, thrombosis, myocardialinfarction, and/or diabetes.

In an embodiment, the disclosure provides a method for the treatment ofautoimmune diseases, inflammatory diseases, cancer, vascular diseases,infectious diseases, thrombosis, myocardial infarction, and/or diabetesin a subject in need thereof using a pharmaceutical compositioncomprising an antigen-binding molecule according to the presentdisclosure.

Further provided is a method of producing an antigen-binding moleculesaccording to the present disclosure in a form suitable foradministration in vivo, the method comprising (a) obtaining anantigen-binding molecule by a method according to the presentdisclosure, and (b) formulating said antigen-binding molecule with atleast one pharmaceutically acceptable carrier, whereby a preparation ofantigen-binding molecule is formulated for administration in vivo.

Pharmaceutical compositions according to the present disclosure comprisea therapeutically effective amount of one or more antigen-bindingmolecules according to the present disclosure dissolved in apharmaceutically acceptable carrier.

In an embodiment, the present disclosure provides a kit comprising theantigen-binding molecule according to the present disclosure or apharmaceutical composition comprising the antigen-binding moleculeaccording to the present disclosure.

In an embodiment, the present disclosure provides a kit comprising theantigen-binding molecule according to the present disclosure or apharmaceutical composition comprising the antigen-binding moleculeaccording to the present disclosure, and a package insert comprisinginstructions for administration of a binding molecule according to thepresent disclosure for treating or delaying progression of cancer orreducing or inhibiting tumor growth in a subject in need thereof.

Dosage

For the prevention or treatment of a disease, the appropriate dosage ofan antigen-binding molecule according to the present disclosure willdepend on the type of disease to be treated, the route ofadministration, the body weight of the individual, the particular typeof antigen-binding molecule, the severity and course of the disease,whether the antigen-binding molecule is administered for preventive ortherapeutic purposes, previous or concurrent therapeutic interventions,the individual's clinical history and response to of antigen-bindingmolecule, and the discretion of the attending physician. Anantigen-binding molecule according to the present disclosure is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, 1 μg/kg to 15 mg/kg(e.g. 0.1 mg/kg-10 mg/kg) of an antigen-binding molecule according tothe present disclosure can be an initial dosage for administration tothe individual, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from 1 μg/kg to 100 mg/kg or more, depending on the factorsmentioned above. For repeated administrations over several days orlonger, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage for an antigen-binding molecule according to thepresent disclosure would be in the range from 0.005 mg/kg to 10 mg/kg.In other non-limiting examples, a dose may also comprise 1 μg/kg bodyweight, 5 μg/kg body weight, 10 μg/kg body weight, 50 μg/kg body weight,100 μg/kg body weight, 200 μg/kg body weight, 350 μg/kg body weight, 500μg/kg body weight, 1 mg/kg body weight, 5 mg/kg body weight, 10 mg/kgbody weight, 50 mg/kg body weight, 100 mg/kg body weight, 200 mg/kg bodyweight, 350 mg/kg body weight, 500 mg/kg body weight, to 1000 mg/kg bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of 5 mg/kg body weight to 100 mg/kg body weight, 5 μg/kgbody weight to 500 mg/kg body weight, etc., can be administered, basedon the numbers described above. Thus, one or more doses of 0.5 mg/kg,2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the individual. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that theindividual receives from two to twenty, or e.g. six doses of theantigen-binding molecule). An initial higher loading dose, followed byone or more lower doses may be administered. An antigen-binding moleculeaccording to the present disclosure will generally be used in atherapeutically amount effective to achieve the intended purpose.

Combination Therapies

An antigen-binding molecule according to the present disclosure may beadministered in combination with one or more other therapeutic agents.“Therapeutic agent” encompasses any agent administered to treat asymptom or disease in an individual in need of such treatment. Incertain embodiments, an additional therapeutic agent is animmunomodulatory agent, a cytostatic agent, an inhibitor of celladhesion, a cytotoxic agent, an activator of cell apoptosis, or an agentthat increases the sensitivity of cells to apoptotic inducers. Suchother therapeutic agents are suitably present in combination in amountsthat are effective for the purpose intended. Combination therapiesencompass combined administration (where two or more therapeutic agentsare included in the same or separate compositions), and separateadministration, in which case, administration of an antigen-bindingmolecule according to the present disclosure can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent. An antigen-binding molecule according to the presentdisclosure can also be used in combination with radiation therapy.

Sequences

TABLE 2Amino acid sequence of the extracellular domain of human CD3 epsilonTarget protein SEQ ID NO: [aa] Mature human CD3 1DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEIL epsilon-ECD (1-118)WQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQS GYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

TABLE 3 VH and VL amino acid sequences of CD3 specific antibody “SP34”Antibody Chain SEQ ID NO [aa] SP34 VH 2EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMN WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGN SYVSWFAYWGQGTLVTVSS SP34 VL 3QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL GQ

TABLE 4 VH and VL amino acid sequences of CD3 specific antibody “I2C”Antibody Chain SEQ ID NO: [aa] I2C VH 4EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMN WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNS YISYWAYWGQGTLVTVSS I2C VL 5QTVVTQEPSLTVSPGGIVTLICGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV L

TABLE 5 VH and VL amino acid sequences of CD3 specific antibody “Roche”Antibody Chain SEQ ID NO: [aa] Roche VH 6EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAM NWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRH GNFGNSYVSWFAYWGQGTLVTVSS Roche VL 7QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNY ANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALVVYSNLWVFG GGTKLTVLGQ

TABLE 6VH and VL amino acid sequence of HER2 specific antibody TrastuzumabAntibody Chain SEQ ID NO: [aa] Trastuzumab VH 8QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY IHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSS Trastuzumab VL 9DIQMTQSPSSLSASVGDRVTITCRASQDVNTAV AWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIK

TABLE 7 Amino acid sequences of peptide linkers SEQ ID Linker NO: [aa](GGS)₃ Linker 10 GGSGGSGGS CH1_(trunc. )Linker 11 ASTKGPCLλ_(trunc. )Linker 12 QPKAAP Hinge_(trunc )Linker 13 DKTHTCPPCPCombined (CH1_(trunc. )+ 14 ASTKGPDKTHTCPPCP Hinge_(trunc.)) LinkerCombined (CLλ_(trunc )+ 15 QPKAAPDKTHTCPPCP Hinge_(trunc.)) Linker

TABLE 8 Amino acid sequences of heterodimeric Fc region subunitsincluding N-terminal located IgG hinge derived linker Linker + Fc SEQregion ID subunits NO: [aa] Linker_((Hinge))- 16DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTC CH2_((AEASS))-VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY CH3_((knob))RVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKLinker_((Hinge))- 17 DKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCCH2_((AEASS))- VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY CH3_((hole))RVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE 9 Amino acid sequences of the polypeptides forming a trivalentbispecific antigen-binding molecule according to the presentdisclosure and as shown in FIG. 1B (without a disulfidestabilized second Fv region) with bivalent bindingto HER2 and monovalent binding to CD3 Construct 1 SEQ  (Trastuzumab/I2C)ID NO: [aa] VH_((Trast.))-CH1-Linker_((GGS)3)- 18QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTY VL_((12C))-Linker_((CLλ+hinge))-IHWVRQAPGKGLEWVARIYPTNGYTRYADSVKG CH2_((AEASS))-CH3_((knob))RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSGGSG GSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPAR FSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGQPKAAPDKTHTCPPCPAP EAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGKVH_((Trast.))-CH1-Linker_((GGS)3)- 19 QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYVH_((12C))-Linker_((CH1+hinge))- IHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGCH2_((AEASS))-CH3_((hole)) RFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSCGGSGGSGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVY YCVRHGNFGNSYISYWAYWGQGTLVTVSSASTKGPDKTHTCPPCPAPEAEGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK VL_((Trast ))-CL 20DIQMTQSPSSLSASVGDRVTITCRASQDVNTAV human IgG kappaAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSR SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

TABLE 10 Amino acid sequences of the polypeptides forming a trivalentbispecific antigen-binding molecule according to the presentdisclosure and as shown in FIG. 1B (with a disulfide stabilizedsecond Fv region) with bivalent binding to HER2 andmonovalent binding to CD3 Construct 2 SEQ (Trastuzumab/I2C) ID NO: [aa]VH_((Trast.))-CH1-Linker_((GGS)3)- 21 QVQLVESGGGLVQPGGSLRLSCAASGFNIKVL(G100C)_((I2C))-Linker_((CLλ+hinge))- DTYIHWVRQAPGKGLEWVARIYPTNGYTRYCH2_((AEASS))-CH3_((knob)) ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGOLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSGGSGGSQTVVTQEP SLTVSPGGIVTLTCGSSTGAVISGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLVVYSNRWVFGCGTKLTVLGQQPKAAPDKTHTCPPCP APEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTL PPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKVH_((Trast.))-CH1-Linker_((GGS)3)- 22 QVQLVESGGGLVQPGGSLRLSCAASGFNIKVH(G44C)_((I2C))-Linker_((CH1+hinge))- DTYIHWVRQAPGKGLEWVARIYPTNGYTRYCH2_((AEASS))-CH3_((hole)) ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSGGSGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKCLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSAST KGPDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISK AKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK VL_((Trast.))-CL 23 DIQMTQSPSSLSASVGDRVTITCRASQDVNThuman IgG kappa AVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYT TPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 11 Amino acid sequences of the polypeptides forming a trivalentbispecific antigen-binding molecule as shown in FIG. 1B(without a disulfide stabilized second Fv region) withbivalent binding to HER2 and monovalent binding to CD3 Contruct 3 SEQ(Trastuzumab/Roche) ID NO: [aa] VH_((Trast.))-CH1-Linker_((GGS)3)- 24QVQLVESGGGLVQPGGSLRLSCAASGFNIK VL_((Roche))-Linker_((CLλ+hinge))-DTYIHWVRQAPGKGLEWVARIYPTNGYTRY CH2_((AEASS))-CH3_((knob))ADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCGGSGGSGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWV QEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLW VFGGGTKLTVLGQPKAAPDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK VH_((Trast.))-CH1-Linker_((GGS)3)-25 QVQLVESGGGLVQPGGSLRLSCAASGFNIK VH_((Roche))-Linker_((CH1+hinge))-DTYIHWVRQAPGKGLEWVARIYPTNGYTRY CH2_((AEASS))-CH3_((hole))ADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCGGSGGSGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWV RQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYC VRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPDKTHTCPPCPAPEAEGAPSVFLFPPK PKIDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSC AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK VL_((Trast.))-CL 26DIQMTQSPSSLSASVGDRVTITCRASQDVNT human IgG kappaAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 12 Amino acid sequences of the polypeptides forming atrivalent bispecific antigen-binding molecule as shown inFIG. 1B (without a disulphide stabilized second Fv region)with bivalent binding to HER2 and monovalent binding to CD3. Construct 4SEQ  (Trastuzumab/SP34) ID NO: [aa] VH_((Trast.))-CH1-Linker_((GGS)3)-27 QVQLVESGGGLVQPGGSLRLSCAASGFNIK VL_((SP34))-Linker_((CLλ+hinge))-DTYIHWVRQAPGKGLEWVARIYPTNGYTRY CH2_((AEASS))-CH3_((knob))ADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCGGSGGSGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWV QEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVF GGGTKLTVLGQPKAAPDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK VH_((Trast. ))-CH1-Linker_((GGS)3)- 28QVQLVESGGGLVQPGGSLRLSCAASGFNIK VH_((SP34))-Linker_((CH1+hinge))-DTYIHWVRQAPGKGLEWVARIYPTNGYTRY CH2_((AEASS))-CH3_((hole))ADSVKGRFTISADTSKNTAYLQMNSLRAEDT AVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCGGSGGSGGSEVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWV RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYC VRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPDKTHTCPPCPAPEAEGAPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSC AVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK VL_((Trast.))-CL 29DIQMTQSPSSLSASVGDRVTITCRASQDVNT human IgG kappaAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC

TABLE 13 Amino acid sequences of the polypeptides forming atrivalent bispecific antigen-binding molecule as shown inFIG. 1B (without a disulfide stabilized second Fv region)with bivalent binding to HER2 and monovalent binding to CD3 Construct 5SEQ  (Trastuzumab/Neg. Ctrl.) ID NO: [aa]VH_((Trast.))-CH1-Linker_((GGS)3)- 30 QVQLVESGGGLVQPGGSLRLSCAASGFNIKVL_((Neg. Ctrl))-Linker_((CLλ+hinge))- DTYIHWVRQAPGKGLEWVARIYPTNGYTRYCH2_((AEASS))-CH3_((knob)) ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSGGSGGSDIELTQPPS VSVAPGQTARISCSGDNLPAYTVIWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTA TLTISGTQAEDEADYYCASWDPSSGVVFGGGTKLTVLGQPKAAPDKTHTCPPCPAPEAEG APSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREE MTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSP GKVH_((Trast.))-CHI-Linker_((GGS)3)- 31 QVQLVESGGGLVQPGGSLRLSCAASGFNIKVH_((Neg. Ctrl.))-Linker_((CH1+hinge))- DTYIHWVRQAPGKGLEWVARIYPTNGYTRYCH2_((AEASS))-CH3_((hole)) ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGSGGSGGSQVQLQQSG PGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVK SRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSS ASTKGPDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK VL_((Trast.))-CL 32DIQMTQSPSSLSASVGDRVTITCRASQDVNT human IgG kappaAVAWYQQKPGKAPKLLIYSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC

WORKING EXAMPLES

The following are examples of molecules and methods according to thepresent disclosure. It is understood that various other embodiments maybe practiced, given the general description provided herein.

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. General informationregarding the nucleotide sequences of human immunoglobulins light andheavy chains is given in: Kabat, E. A. et al., (1991) Sequences ofProteins of Immunological Interest, 5th ed., NIH Publication No.91-3242.

Example 1: Preparation, Production and Characterization of TrivalentBispecific Antigen Binding Molecules

The antigen-binding molecules as exemplified herein were built from anaglycosylated monoclonal human IgG1 template antibody incorporating anadditional Fv region (Fv₂) between the Fc region and the two Fab arms ofa regular human IgG1 molecule. A basic structure of such a molecule isprovided in FIG. 1B.

The fusion between the two Fab arms and the Fc region was achieved byusing two glyine-serine linkers ((GGS)₃) (SEQ ID NO: 10) between theC-terminus of the two Fab heavy chains and the N-terminus of thevariable domains (VH₂ and VL₂) of the incorporated additional Fv region.The fusion between the variable domains of the second Fv region (Fv₂)and the two Fc region subunits was achieved by using peptide linkersbuild from the first 5 amino acid residues of a CU. (QPKAAP (SEQ ID NO:12)) or CH1 (ASTKGP (SEQ ID NO: 11) constant domain and a portion of ahuman IgG1 hinge sequence (DKTHTCPPCP (SEQ ID NO: 13). The use of thehuman IgG1 hinge sequence allowed for a further stabilization of theheterodimeric molecule via the formation of interchain-disulfide bridgesbetween the two employed peptide linkers. The Fc region was modified byintroducing mutations into the CH3 domain of each Fc region subunitaccording to the “knob-into-holes” technology. Thereby, the polypeptidecomprising one mutated CH3 domain is forced to heterodimerize with theother polypeptide comprising the other CH3 domain, which is engineeredin a complementary manner.

In the below exemplified bispecific trivalent constructs, the two Fabs(Fab₁ and Fab₂) present in the two Fab arms of the antigen-bindingmolecule, specifically bind to the cancer associated target HER2 in abivalent manner, whereas the incorporated second Fv region (Fv₂)specifically binds to human CD3 epsilon in a monovalent fashion.

For HER2 binding, nucleotide sequences encoding the VH and VL domainsfrom “Trastuzumab” (HERCEPTIN®) as described by Baselga et al. 1998,Cancer Res 58(13): 2825-2831) were used. Trastuzumab and its method ofpreparation are described in U.S. Pat. No. 5,821,337.

For CD3 binding, the following nucleotide sequences encoding the VH andVL domains of the following CD3 antibodies were used:

-   -   SP34: a monoclonal antibody described by Yoshino et al. (Exp.        Anim. 49(2), 97-110, 2000).    -   I2C: a monoclonal antibody described in WO 2008/119566 (MICROMET        AG)    -   A monoclonal CD3 antibody disclosed in WO 2016/020309 (F.        HOFFMANN-LA ROCHE AG) referred herein to as antibody “Roche”.    -   An in-house negative control antibody with specificity for        chicken lysozyme.

A summary of the individual components (Fabs, Linkers, Fv regions, Fcregion etc.) of the produced bispecific trivalent antigen-bindingmolecules according to the present disclosure (referred herein to asConstructs 1-5) made in accordance with the examples described hereinare set forth in Tables: 3-13.

Gene Synthesis

All nucleic acid sequences or desired gene segments were eithergenerated by PCR using appropriate templates or were gene synthesized aslinear DNA fragments with appropriate flanking regions (e.g. suitablerestriction enzyme recognition sites, linker sequences) in-house or byan external provider. The nucleic acid sequences or gene segmentsflanked by singular restriction endonuclease cleavage sites were clonedinto respective expression vectors (e.g. mammalian IgG expressionvectors) or sequencing vectors using standard molecular biology methods.When intended for use in mammalian expression vectors, all constructswere designed with a 5′-end DNA sequence coding for a leader peptidewhich targets proteins for secretion in eukaryotic cells. The DNAsequence of the subcloned gene fragments was confirmed by DNA by doublestrand sequencing.

Production

For expression of Construct 1-5, exponentially growing eukaryotic HEK293cells were transfected with a mammalian two vector expression systemencoding all components of the Constructs, resulting in a 1:1:2 ratio ofthe two polypeptides comprising the Fc region subunits and thepolypeptide comprising the light chain of the first and second Fab,respectively.

Cell culture supernatants were harvested on day 6 post transfection andsubjected to standard Protein A affinity chromatography (MabSelectSURE|GE Healthcare). Buffer exchange was performed to 1× Dulbcecco's PBS(pH 7.2|Invitrogen) and samples were sterile filtered (0.2 μm poresize). Protein concentrations were determined by UV-spectrophotometryand purities of the constructs were analyzed under denaturing, reducingand non-reducing conditions using CE-SDS (LabChip GXII|PerkinElmer|USA). HP-SEC was performed to analyze IgG preparations in nativestate.

Production Results

Table 14 summarizes yields and final monomer content of the differentpreparations obtained for the produced constructs. In general, theconstructs could be generated by the described production andpurification method with yields between 29-75 mg/L and final monomercontent between 65-90%. The use of a stabilizing disulfide bridge(VH-G44C/VL-G100C) between the VH and VL domain of the second Fv regionin Construct 2 resulted in a significant reduced yield and monomercontent when compared to corresponding Construct 1 lacking thestabilizing disulfide bridge.

TABLE 14 Yields and final monomer content. Volu- metric Monomer YieldContent Construct SEQ ID NOs: [mg/L] [%] Contruct 1 Trastuzumab/I2C 18,19, 20  62*  88* Construct 2 Trastuzumab/I2C 21, 22, 23 29 65(VH-G44C/VL- G100C) Contruct 3 Trastuzumab/Roche 24, 25, 26 75 90Contruct 4 Trastuzumab/SP34 27, 28, 29 53 90 Contruct 5 Trastuzumab/Neg.30, 31, 32 69 90 Ctrl. *average of n = 2

Example 2: Binding of Trivalent Bispecific Antigen-Binding Molecules toCD3 and HER2 Expressed on Cells Target Cells:

For the assessment of HER2 targeting of the bispecific trivalentConstructs 1, 3, 4 and 5 with bivalent binding to HER2 and monovalentbinding to CD3, the following tumor cell lines were used: the HER2positive human adenocarcinoma SKOV-3 [SKOV3] (ATCC® HTB-77™) cell lineand the HER2 negative human adenocarcinoma MDA-MB-468 (ATCC® HTB-132™)cell line. In addition, a human CD3 positive T cell leukemia cell line,Jurkat (ATCC #TIB-152) was used to assess binding to human CD3.

Method:

Jurkat or SKOV-3 cells were resuspended and counted in wash buffer (DPBSwith calcium and magnesium (Gibco, #14040174) supplemented with 3% FBSand 0.02% sodium acid). 6E+04 cells per well were seeded in 384 wellV-bottom plates (Greiner bio-one, #781280) and incubated with seriallydiluted constructs (titration range 500 nM to 0.5 nM) for 1 h on ice.Cells were washed 2 times in wash buffer. Bound constructs were detectedusing AlexaFluor647-conjugated detection antibody directed against humanF(ab′)₂ fragment (Jackson Immuno Research, #109-606-097). Constructstaining was measured using IntelliCyt iQue flow cytometer and analyzedin or ForeCyt (version 4.1.5379, IntelliCyt) software. EC₅₀ values werecalculated using 4-parameter non-linear regression analysis in Prismsoftware (GraphPad Software Inc., version 5.04).

Results:

Results of the experiment are summarized in Table 15 and FIG. 3A (Jurkatcells) and FIG. 3B (SKOV-3 cells) and reveal that the bispecifictrivalent Constructs 1, 3 and 4 specifically bind to HER2 and CD3expressed on cells in a dose dependent manner. Furthermore, no bindingto the HER2 and CD3 negative cell line is observable for the testedconstructs (data not shown). Negative control Construct 5 shows nobinding activity using either cell line.

TABLE 15 Cell binding of HER2 × CD3 bispecific constructs to HER2expressing SKOV-3 cells FACS EC₅₀ [nM] Construct SEQ ID NOs: SKOV-3Construct 1 Trastuzumab/I2C 18, 19, 20 2.41 Construct 3Trastuzumab/Roche 24, 25, 26 3.72 Construct 4 Trastuzumab/SP34 27, 28,29 4.17 Construct 5 Trastuzumab/Neg. Ctrl. 30, 31, 32 3.96

Example 3: Reporter Gene Assay—Testing of Trivalent BispecificAntigen-Binding Molecules on SKOV-3 and Jurkat Cells Transfected withthe NFAT Reporter Gene Target and Effector Cells:

For the evaluation of the functional activity of the bispecifictrivalent Constructs 1, 3, 4 and 5, Jurkat cells (ATCC #TIB-152)transiently transfected with an NFAT reporter gene construct were usedas surrogate effector cells. As target cells the following tumor celllines were used: the HER2 positive human adenocarcinoma SKOV-3 [SKOV3](ATCC® HTB-77™) cell line and the HER2 negative human adenocarcinomaMDA-MB-468 (ATCC® HTB-132™) cell line.

The following growth media were used for maintenance of the cell lines:(a) Jurkat: RPMI-1640+L-Glutamine (Thermo Fisher, #21875-034)supplemented with 10% FCS (Sigma, #F7524); (b) SKOV-3: McCoys 5a(ThermoFisher, #10938), supplemented with 10% FCS (Sigma #F7524) (c)MDA-MB-468: DMEM-L-Glutamine (ThermoFisher, #10938) supplemented with 1×GlutaMAX™ (ThermoFisher, #35050-061), 1× Sodium Pyruvate (ThermoFisher,#11360-039) and 10% FCS (Sigma #F7524).

Method:

SKOV-3 and MDA-MB-468 cells were diluted in growth medium to a densityof 4E+05 cells/ml. 100 μl cell suspension corresponding to 40,000 cellswere seeded in each well of a tissue culture treated 96 well plate(Corning, #3917) and incubated overnight in a humidified incubator at37° C. and 5% CO₂. Jurkat cells were resuspended in growth medium to aconcentration of 2.5E+05 cells/ml. Transfection componentspGL4.30[luc2P/N FAT-RE/Hygro] reporter gene vector, OptiMEM-I medium(Life Technologies, #31985-047) and TransIT-LT1 transfection reagent(Mirus, #MIR2304) were incubated for 15 min at RT, then added to theJurkat cell suspension and incubated for 17 h in a humidified incubatorat 37° C. and 5% CO₂. Jurkat cells were harvested and resuspended ingrowth medium at a concentration of 1.2E+06/ml. Medium was removed fromcoated target cells and replaced by 50 μl Jurkat cell suspensioncorresponding to 60,000 cells per well. Constructs 1, 3, 4 and negativecontrol Construct 5 were serially diluted in Jurkat growth medium. 50 μlconstruct dilution was added to each well resulting in a finalconcentration range of 31 nM to 0.12 nM. Assay plates were incubated for5 h in a humidified incubator at 37° C. and 5% CO₂. Bright-Glo™ Reagent(Promega, #E2620) was reconstituted according to manufacturer'sinstructions. Assay plates and reagent were equilibrated at roomtemperature. 100 μl of the Bright-Glo™ reagent was added to each well ofthe assay plate and mixed. Luminescence was measured using anInfiniteM1000 Pro plate reader (Tecan).

Results:

The results of the experiments are summarized in Table 16 and FIG. 4A(SKOV-3 cells) and FIG. 4B (MDA-MB-468 cells). Constructs 1, 3, 4 inducedose-dependent luciferase activity in the presence of the HER2expressing target cell line SKOV-3 with EC₅₀ concentrations ranging from0.7 nM to 1.8 nM. Construct 1 shows the highest efficacy and potency asindicated by EC₅₀ and maximum luciferase activity level, respectively.In the presence of HER2-negative MDA-MB-468 target cells, Construct 1induces weak luciferase activity at high concentrations. Constructs 3, 4are inactive in the absence of HER2. Negative control Construct 5 showsno activity using either target cell line.

TABLE 16 Induction of luciferase activity of HER2 × CD3 bispecificconstructs in the presence of SKOV-3 cells. EC₅₀ [nM] Construct SEQ IDNOs: SKOV-3 Construct 1 Trastuzumab/I2C 18, 19, 20 0.71 Construct 3Trastuzumab/Roche 24, 25, 26 1.53 Construct 4 Trastuzumab/SP34 27, 28,29 1.75 Construct 5 Trastuzumab/Neg. Ctrl. 30, 31, 32 no activity

Example 4: Re-Directed T-Cell Cytotoxicity Mediated by TrivalentBispecific Antigen-Binding Molecules

Bispecific trivalent Constructs 1, 3, 4 and 5 were analyzed for theirpotential to induce T-cell-mediated killing of tumor cells upon bindingto CD3 and HER2.

Method

Human whole blood from healthy donors was collected in Li-Heparincontaining S-Monovette containers (Sarstedt). Blood was transferred to50 ml conical tubes and mixed with an equal volume of PBS containing 2%fetal bovine serum (Sigma, #F7524) and 2 mM EDTA. Diluted blood wastransferred to SepMate-50 tubes (StemCell Technologies, #86450)containing 15 ml Biocoll solution (Biochrom, #L6115) and centrifuged for10 min at 1200×g. Supernatant was transferred into a 50 ml conical tube,diluted to 45 ml with PBS and centrifuged for 8 min at 300×g.Supernatant was discarded, cell pellet resuspended in 1 ml PBS and cellscounted using a Neubauer chamber.

5,000 HER2 expressing SKBR3 cells and HER2 negative MDA-MB-468 cellswere suspended in culture medium (SKBR3: McCoy's 5A Medium(ThermoFisher, #26600), 10% FCS (Sigma, #F7524); MDA-MB-468: DMEM(ThermoFisher, #10938), 1× GlutaMAX™ (ThermoFisher, #35050-061), 1×Sodium Pyruvate (ThermoFisher, #11360-039), 10% FCS seeded in black 96well assay plates (Corning, #3340) and incubated over night at 37° C.and 5% CO₂. CellToxGreen dye (Promega, #G8731), bispecificantigen-binding molecules diluted to 100 nM and 100,000 purified PBMCs,all diluted in assay medium comprising RPMI 1640 w/o Phenol red (Gibco,#32404-014), GlutaMAX and 10% fetal bovine serum, were added to thecells and incubated for 48 h at 37° C. and 5% CO₂. Cytotoxic activitywas assessed by measuring incorporated CellToxGreen fluorescence at 485nm excitation and 535 nm emission using a Tecan Infinite F500 device.

Results:

The results of the experiments are shown in FIG. 5. Co-cultivation ofPBMCs with Construct 1, 3 and 4 induces killing of HER2 expressing SKBR3target cells at a tested construct concentration of 100 nM. In theabsence of the HER2 negative MDA-MB-468 cells, none of the testedconstructs stimulates cytotoxic activity. Negative control Construct 5induces no activity using either target cell line.

Example 5: Affinity Determination Methods

Kinetic characterization of the interaction between human CD3 epsilonand an antigen-binding molecule with monovalent specificity for CD3 andbivalent specific for HER2 (Construct 1 and 3) was carried out inantigen-binding molecule capture format, with the antigen being appliedas analyte in solution. High-capacity capture surfaces were prepared byloading biotinylated MabSelect SuRe ligand (non-biotinylated ligand: GEHealthcare, 28-4018-60) onto several streptavidin sensors (fortébio,part 18-5021). Each cycle of the kinetic experiment consisted of capturesteps (of one ligand on several sensors used in parallel), followed byan analyte binding step (association phase, different analyteconcentrations and assay buffer, i.e. antigen concentration 0 for blanksubtraction). After binding, the dissociation of bound antigen wasmonitored (sensors exposed to assay buffer). At the end of each cycle,bound ligand and/or ligand-antigen complex was removed from the sensorsurfaces by 2 consecutive regeneration steps à 20 s with 10 mMGlycine/HCl pH1.5 (GE Healthcare, BR 100354), while maintaining theintegrity of the capture surface.

Signals recorded on the sensor with captured ligand, but exposed toassay buffer instead of antigen during binding were subtracted from thesensorgrams with non-zero antigen concentrations to correct e.g.potential dissociation of captured ligand. Association was recorded for300 s and dissociation for 300 s at an orbital shaking speed of 1000rpm. DPBS (GIBCO, no Ca²⁺, no Mg²⁺; Thermo Fisher Cat. No. 14190)supplemented with 0.05% (v/v) Polysorbate 20 (Merck, 8.22184.0500) and0.1% (w/v) bovine serum albumin (Sigma, A7906) was used as assay buffer.Capture levels of ligands were adjusted to approx. 2 nm to achievesaturation levels R max of approx. 0.3 nm by the hCD3e analyte. Sevendifferent analyte concentrations were used for analysis during kineticexperiments (pMAX_hCD3e(1-118)_F-chLys_avi; applied molarities15.625-1000 nM, in a 2-fold serial dilution series).

Sensorgrams were evaluated with Data Analysis Software v 10(Octet/fortébio). All sensorgrams were fitted to a 1:1 binding model todetermine k_(on) and k_(off) rate constants, which were used tocalculate the K_(D) value. For kinetic profiles deviating from theexpected 1:1 binding, the sensorgrams were evaluated using a bestapproximation to the monovalent kinetics, and results marked withcomment “heterogeneous binding”. These results are considered lessprecise than kinetic profiles completely following the expectedmonovalent binding kinetics, but are assumed to be good approximationsfor K_(D). Additionally, extrapolated saturation levels (R max) were setinto relation with the obtained capture levels of ligand, taking intoaccount the respective molecular weights of ligand and CD3 protein toassess if the observed binding events could be explained with theexpected stoichiometry and monovalent binding.

Results: Binding to Human CD3 Epsilon

The results of the experiments are summarized in Table 17. The observedbinding was used to extrapolate the saturation level of CD3, and setinto relation to the capture level of ligand. The experimentalsaturation R max was found within in the range (100±15%) theoreticallyexpected for monovalent binding of CD3 to a fully active, monovalentantibody.

The results reveal that the relative position of the second Fv regionwith specificity for CD3 is not detrimental to the binding activity ofthe used CD3 specific antibody.

TABLE 17 Affinities of bispecific trivalent Construct 1 and Construct 3with monovalent binding to human CD3 epsilon SEQ ID kon koff KDConstruct NO: [1/Ms] [1/s] [nM] Comment Construct 1 Trastuzumab/ 18, 19,4.57E+05 4.47E−04 1.0 slightly heterogenous I2C 20 binding Construct 3Trastuzumab/ 24, 25, 5.19E+05 3.24E−03 6.2 — Roche 26

1. An antigen-binding molecule, comprising a) a first Fab comprising afirst Fv region, which specifically binds to a first antigen, b) asecond Fv region which specifically binds to a second antigen and c) asecond Fab comprising a third Fv region, which specifically binds to athird antigen, and d) a Fc region composed of a first and second Fcregion subunit; wherein i. the C-terminus of the heavy or light chain ofthe first Fab is fused to the N-terminus of the VH or VL of the secondFv region, and wherein ii. the C-terminus of the VH or VL of the secondFv region is fused to the N-terminus of the first Fc region subunit andthe N-terminus of the second Fc subunit is fused to the C-terminus ofthe complementary variable domain of the second Fv region, and whereiniii. the C-terminus of the heavy or light chain the second Fab is fusedto the N-terminus of the VH or VL of the second Fv region with theproviso that the first and second Fab are fused to distinct variabledomains of the second Fv region, and wherein iv. in the CH3 domain ofthe first Fc region subunit, the threonine residue at position 366 isreplaced with a tryptophan residue (T366W) and the serine residue atposition 354 is replaced with a cysteine residue (S354C) and in the CH3domain of the second Fc region subunit the tyrosine residue at position407 is replaced with a valine residue (Y407V), the threonine residue atposition 366 is replaced with a serine residue (T366S), the leucineresidue at position 368 is replaced with an alanine residue (L368A) andthe tyrosine residue at position 349 is replaced by a cysteine residue(Y349C) with numbering according EU index.
 2. The antigen-bindingmolecule according to claim 1, wherein each fusion occurs via a peptidelinker.
 3. The antigen-binding molecule according to claim 1, whereinthe antigen-binding molecule is composed of at least 4 polypeptides,wherein a. a first polypeptide comprises the light or heavy chain of thefirst Fab, b. a second polypeptide comprises from its N-terminus to itsC-terminus i. the complementary light or heavy chain of the first Fab,ii. the VH or VL of the second Fv region and iii. the first or second Fcregion subunit c. a third polypeptide comprises from its N-terminus toits C-terminus i. the light or heavy chain of the second Fab, ii. thecomplementary VH or VL of the second Fv region and iii. thecomplementary first or second Fc region subunit d. a fourth polypeptidecomprises the complementary light or heavy chain of the second Fab. 4.The antigen-binding molecule according to claim 1, wherein the thirdantigen is identical to the first antigen.
 5. The antigen-bindingmolecule according to claim 1, wherein the antigen-binding moleculeprovides bivalent binding to the first antigen and monovalent binding tothe second antigen.
 6. The antigen-binding molecule according to claim 1wherein the antigen-binding molecule is a trivalent bispecificantigen-binding molecule.
 7. The antigen-binding molecule according toclaim 1, wherein the second antigen is expressed on an immune effectorcell.
 8. The antigen-binding molecule according to claim 1, wherein thefirst antigen is a member of the T-cell receptor complex.
 9. Theantigen-binding molecule according to claim 1, wherein the secondantigen is CD3.
 10. The antigen-binding molecule according to claim 1,wherein the Fc region is a human IgG1 Fc region.
 11. The antigen-bindingmolecule according to claim 1, wherein the Fc region comprises one ormore amino acid modification promoting the association of the first andsecond Fc region subunit.
 12. The antigen-binding molecule according toclaim 1, wherein in each of the Fc region subunit at least 5 amino acidresidues in the positions corresponding to positions L234, L235, G237,A330, P331 with numbering according EU index in a human IgG1 are mutatedto A, E, A, S, and S, respectively.
 13. A pharmaceutical compositioncomprising the antigen-binding molecule according to claim 1 and apharmaceutically acceptable carrier or excipient.
 14. (canceled)
 15. Amethod for re-directing cytotoxic activity of a T-cell to a cancer cellcomprising contacting said cancer cell in the presence of a T-cell witha antigen-binding molecule according to claim
 1. 16. A method fortreating a disease in an individual, said method comprisingadministering an effective amount of the pharmaceutical composition ofclaim 13 to the individual.
 17. The method of claim 16 wherein thedisease is an autoimmune disease, an inflammatory disease, cancer, avascular disease, an infectious disease, thrombosis, myocardialinfarction or diabetes.
 18. The method of claim 16 wherein the diseaseis a proliferative disease.
 19. The method of claim 16 wherein thedisease is cancer.